Genetically modified cells, tissues, and organs for treating disease

ABSTRACT

Genetically modified cells, tissues, and organs for treating or preventing diseases are disclosed. Also disclosed are methods of making the genetically modified cells and non-human animals.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.14/965,451, filed Dec. 10, 2015, entitled “Genetically Modified Cells,Tissues, and Organs for Treating Disease,” which claims the benefit ofU.S. Provisional Patent Application No. 62/090,037, filed on Dec. 10,2014, and U.S. Provisional Patent Application No. 62/253,493, filed onNov. 10, 2015, which are both herein incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 10, 2017, isnamed 47190-701.201_SL.txt and is 713,905 bytes in size.

BACKGROUND OF THE DISCLOSURE

There is a shortage of organs, tissues or cells available fortransplantation in recipients such as humans. Xenotransplantation orallotransplantation of organs, tissues, or cells into humans has thepotential to fulfill this need and help hundreds of thousands of peopleevery year. Non-human animals can be chosen as organ donors based ontheir anatomical and physiological similarities to humans. Additionally,xenotransplantation has implications not only in humans, but also inveterinary applications.

However, unmodified wild-type non-human animal tissues can be rejectedby recipients, such as humans, by the immune system. Rejection isbelieved to be caused at least in part by antibodies binding to thetissues and cell-mediated immunity leading to graft loss. For example,pig grafts can be rejected by cellular mechanisms mediated by adaptiveimmune cells.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications herein areincorporated by reference to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated by reference. In the event ofa conflict between a term herein and a term in an incorporatedreference, the term herein controls.

SUMMARY

Disclosed herein are compositions and methods for treating or preventingdiseases. Also disclosed are genetically modified cells and methods ofmaking the genetically modified cells for treating or preventingdisease. Further disclosed are genetically modified non-human animalsand methods of making genetically modified non-human animals that can beused in treating or preventing disease, e.g., by later extracting cells,tissues, or organs from these genetically modified non-human animals andtransplanting them into a subject. Also disclosed herein are methods fortreating or preventing diseases using the genetically modified cells,tissues, and organs. Additionally disclosed are methods for treating orpreventing diseases using cells, tissues, and/or organs from geneticallymodified non-human animals.

In one aspect, disclosed herein is a genetically modified animal withreduced protein expression of one or more first genes, where thegenetically modified animal is a member of the Laurasiatheria superorderor is a non-human primate, where the one or more first genes comprise a)a component of a major histocompatibility complex (MHC) I-specificenhanceosome, b) a transporter of an MHC I-binding peptide, and/or c)complement component 3 (C3), where the reduced protein expression is incomparison to a non-genetically modified counterpart animal. In somecases, the member of the Laurasiatheria super order is an ungulate. Insome cases, the ungulate is a pig. In some cases, the protein expressionof the one or more first genes is absent in the genetically modifiedanimal. In some cases, the reduction of protein expression inactivates afunction of the one or more first genes. In some cases, the geneticallymodified animal has reduced protein expression of two or more the firstgenes. In some cases, the genetically modified animal comprises reducedexpression of a component of a MHC I-specific enhanceosome, where thecomponent of a MHC I-specific enhanceosome is NOD-like receptor familyCARD domain containing 5 (NLRC5). In some cases, the geneticallymodified animal comprises reduced expression of a transporter of a MHCI-binding peptide, where the transporter is transporter associated withantigen processing 1 (TAP1). In some cases, the genetically modifiedanimal comprises reduced expression of comprising C3. In some cases, thegenetically modified animal has reduced protein expression of three ormore the first genes.

In some cases, the genetically modified animal further comprises reducedprotein expression of one or more second genes, where the one or moresecond genes comprise: a) a natural killer (NK) group 2D ligand, b) anendogenous gene not expressed in a human, c) a CXC chemokine receptor(CXCR) 3 ligand, and/or d) MHC II transactivator (CIITA), where thereduced protein expression is in comparison to a non-geneticallymodified counterpart animal. In some cases, the protein expression ofthe one or more second genes is absent in the genetically modifiedanimal. In some cases, the reduction of protein expression inactivates afunction of the one or more second genes. In some cases, the geneticallymodified animal comprises reduced protein expression of a NK group 2Dligand, where the NK group 2D ligand is MHC class I polypeptide-relatedsequence A (MICA) or MHC class I polypeptide-related sequence B (MICB).In some cases, the genetically modified animal comprises reduced proteinexpression of an endogenous gene not expressed in a human, where theendogenous gene not expressed in a human is glycoproteingalactosyltransferase alpha 1,3 (GGTA1), putative cytidinemonophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH),or β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). In some cases, thegenetically modified animal comprises reduced protein expression of aCXCR3 ligand, where the CXCR3 ligand is C-X-C motif chemokine 10(CXCL10).

In some cases, the genetically modified animal further comprises one ormore exogenous polynucleotides encoding one or more proteins orfunctional fragments thereof, where the one or more proteins comprise:a) an MHC I formation suppressor, b) a regulator of complementactivation, c) an inhibitory ligand for NK cells, d) a B7 family member,e) CD47, f) a serine protease inhibitor, and/or g) galectin. In somecases, the one or more proteins are human proteins. In some cases, thegenetically modified animal comprises one or more exogenouspolynucleotides encoding an MHC I formation suppressor, where the MHC Iformation suppressor is infected cell protein 47 (ICP47). In some cases,the genetically modified animal comprises one or more exogenouspolynucleotides encoding a regulator of complement activation, where theregulator of complement activation is cluster of differentiation 46(CD46), cluster of differentiation 55 (CD55), or cluster ofdifferentiation 59 (CD59). In some cases, the genetically modifiedanimal comprises one or more exogenous polynucleotides encoding aninhibitory ligand for NK cells, where the inhibitory ligands for NKcells is leukocyte antigen E (HLA-E), human leukocyte antigen G (HLA-G),or β-2-microglobulin (B2M). In some cases, the genetically modifiedanimal comprises one or more exogenous polynucleotides encoding HLA-G,where the HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, orHLA-G7. In some cases, the HLA-G is HLA-G1. In some cases, thegenetically modified animal comprises one or more exogenouspolynucleotides encoding a B7 family member, where the B7 family memberis a programed death-ligand. In some cases, the programed death-ligandis programed death-ligand 1 (PD-L1) or programed death-ligand 2 (PD-L2).In some cases, the one or more exogenous polynucleotides encode bothPD-L1 and PD-L2. In some cases, the genetically modified animalcomprises one or more exogenous polynucleotides encoding a serineprotease inhibitor, where the serine protease inhibitor is serineprotease inhibitor 9 (Spi9). In some cases, the genetically modifiedanimal comprises one or more exogenous polynucleotides encoding agalectin, where the galectin is galectin-9.

In some cases, the genetically modified animal comprises reduced proteinexpression of NLRC5 or TAP1, C3, reduced protein expression of CXCL10,GGTA1, CMAH, and/or B4GALNT2; and/or one or more exogenouspolynucleotides encoding HLA-G1, HLA-E, or a functional fragmentthereof, PD-L1 or a functional fragment thereof, PD-L2 or a functionalfragment thereof, and/or CD47 or a functional fragment thereof. In somecases, the one or more exogenous polynucleotides are inserted adjacentto a ubiquitous promoter. In some cases, the ubiquitous promoter is aRosa26 promoter. In some cases, the one or more exogenouspolynucleotides are inserted adjacent to a promoter of a targeted geneor within the targeted gene. In some cases, the targeted gene is one ofthe first genes or one of the second genes. In some cases, the proteinexpression of the one or more first genes is reduced using a CRISPR/cassystem. In some cases, the protein expression of the one or more secondgenes is reduced using a CRISPR/cas system.

In another aspect, disclosed herein is a genetically modified animalthat is a member of the Laurasiatheria superorder or is a non-humanprimate comprising: an exogenous polynucleotide encoding an inhibitoryligand for an NK cell or a functional fragment thereof, and reducedprotein expression of an endogenous gene, where the reduced proteinexpression is in comparison to a non-genetically modified counterpartanimal. In some cases, the inhibitory ligand for an NK cell is HLA-E orHLA-G. In some cases, the inhibitory ligand for an NK cell is HLA-G,where the HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, orHLA-G7. In some cases, the HLA-G is HLA-G. In some cases, the endogenousgene is a gene not expressed in a human. In some cases, the endogenousgene is GGTA1, CMAH, and/or B4GALNT2.

In some cases, the genetically modified animal further comprisesexogenous polynucleotides encoding: a) PD-L1 or a functional fragmentthereof, b) PD-L2 or a functional fragment thereof, and/or c) CD47 or afunctional fragment thereof. In some cases, the exogenouspolynucleotides are inserted adjacent to a ubiquitous promoter. In somecases, the ubiquitous promoter is a Rosa26 promoter. In some cases, theexogenous polynucleotides are inserted adjacent to a promoter of theendogenous gene, or within the endogenous gene. In some cases, theprotein expression of the endogenous genes is reduced using a CRISPR/cassystem.

Further disclosed herein is a population of genetically modified animalscomprising two or more animals disclosed in the application. In somecases, at least two or more animals have identical phenotypes. In somecases, at least two or more animals have identical genotypes.

In another aspect, disclosed herein is a genetically modified cell froma member of the Laurasiatheria superorder or a non-human primate,comprising reduced protein expression of one or more first genes, wherethe one or more first genes comprise: a) a component of a MHC I-specificenhanceosome, b) a transporter of a MHC I-binding peptide, and/or c) C3,where the reduced protein expression is in comparison to anon-genetically modified counterpart cell. In some cases, thegenetically modified cell comprises reduced protein expression of acomponent of a MHC I-specific enhanceosome, where the component of MHCI-specific enhanceosome is NLRC5. In some cases, the geneticallymodified cell comprises reduced protein expression of a transporter of aMHC I-binding peptide, where the transporter of a MHC I-binding peptideis TAP1. In some cases, the genetically modified cell comprises reducedprotein expression of C3.

In some cases, the genetically modified cell further comprises reducedprotein expression of one or more second genes, where the one or moresecond genes comprise: a) an NK group 2D ligands, b) an endogenous genenot expressed in a human, c) a CXCR3 ligand, and/or d) CIITA, where thereduced protein expression is in comparison to a non-geneticallymodified counterpart cell. In some cases, the genetically modified cellcomprises reduced protein expression of an NK group 2D ligand, where theNK group 2D ligand is MICA and/or MICB. In some cases, the geneticallymodified cell comprises reduced protein expression of an endogenous genenot expressed in a human, where the endogenous gene not expressed in ahuman is GGTA1, CMAH, and/or B4GALNT2. In some cases, the geneticallymodified cell comprises reduced protein expression of a CXCR3 ligand,where the CXCR3 ligand is CXCL10.

In some cases, the genetically modified cell further comprises one ormore exogenous polynucleotides encoding one or more proteins orfunctional fragments thereof, where the one or more proteins orfunctional fragments thereof comprise: an MHC I formation suppressor, aregulator of complement activation, an inhibitory ligand for NK cells, aB7 family member, CD47, a serine protease inhibitor, and/or galectin. Insome cases, the one or more proteins or functional fragments thereof arehuman proteins. In some cases, the genetically modified cell comprisesone or more exogenous polynucleotides encoding an MHC I formationsuppressor, where the MHC I formation suppressor is ICP47. In somecases, the genetically modified cell comprises comprising one or moreexogenous polynucleotides encoding a regulator of complement activation,where the regulator of complement activation is CD46, CD55, and/or CD59.In some cases, the genetically modified cell comprises one or moreexogenous polynucleotides encoding an inhibitory ligand for NK cells,where the inhibitory ligands for NK cells is HLA-E, HLA-G, and/or B2M.In some cases, the genetically modified cell comprises the inhibitoryligands for NK cells is HLA-G, and the HLA-G is HLA-G1, HLA-G2, HLA-G3,HLA-G4, HLA-G5, HLA-G6, and/or HLA-G7. In some cases, the geneticallymodified cell comprises one or more exogenous polynucleotides encoding aB7 family member, where the B7 family member is a programeddeath-ligand. In some cases, the HLA-G is HLA-G1. In some cases, theprogramed death-ligand is programed death-ligand 1 (PD-L1) and/orprogramed death-ligand 2 (PD-L2). In some cases, the programeddeath-ligand is both PD-L1 and PD-L2. In some cases, the geneticallymodified cell comprises one or more exogenous polynucleotides encoding aserine protease inhibitor, where the serine protease inhibitor is serineprotease inhibitor 9 (Spi9). In some cases, the genetically modifiedcell comprises one or more exogenous polynucleotides encoding galectin,where the galectin is galectin-9.

In some cases, the genetically modified cell comprises reduced proteinexpression of NLRC5 or TAP1, C3, CXCL10, GGTA1, CMAH, and/or B4GALNT2;and/or exogenous polynucleotides encoding i) HLA-G1, HLA-E, or afunctional fragment thereof, ii) PD-L1 or a functional fragment thereof,iii) PD-L2 or a functional fragment thereof, and/or iv) CD47 or afunctional fragment thereof. In some cases, the one or more exogenouspolynucleotides are inserted adjacent to a ubiquitous promoter. In somecases, the ubiquitous promoter is a Rosa26 promoter. In some cases, theone or more exogenous polynucleotides are inserted adjacent to apromoter of a targeted gene or within the targeted gene. In some cases,the targeted gene is one of the first genes or one of the second genes.In some cases, the protein expression of the one or more first genes isreduced using a CRISPR/cas system. In some cases, the protein expressionof the one or more second genes is reduced using a CRISPR/cas system.

In another aspect, disclosed herein is a genetically modified cell froma member of the Laurasiatheria superorder or a non-human primate,comprising: a) an exogenous polynucleotide encoding an inhibitory ligandfor an NK cell or a functional fragment thereof, and b) reduced proteinexpression of an endogenous gene, where the reduced protein expressionis in comparison to a non-genetically modified counterpart cell.

In some cases, the inhibitory ligand for an NK cell is HLA-E or HLA-G.In some cases, the inhibitory ligand for an NK cell is HLA-G, and theHLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7. Insome cases, the HLA-G is HLA-G1. In some cases, the endogenous gene isnot expressed in a human. In some cases, the endogenous gene is GGTA1,CMAH, and/or B4GALNT2. In some cases, the genetically modified cellfurther comprises exogenous polynucleotides encoding: a) PD-L1 or afunctional fragment thereof, b) PD-L2 or a functional fragment thereof,and/or c) CD47 or a functional fragment thereof. In some cases, theexogenous polynucleotides are inserted adjacent to a ubiquitouspromoter. In some cases, the ubiquitous promoter is a Rosa26 promoter.In some cases, the exogenous polynucleotides are inserted adjacent to apromoter of the endogenous gene, or within the endogenous gene. In somecases, the protein expression of the endogenous genes is reduced using aCRISPR/cas system. In some cases, the genetically modified cell is apancreatic, kidney, eye, liver, small bowel, lung, or heart cell. Insome cases, the genetically modified cell is a pancreatic islet cell. Insome cases, the pancreatic islet cell is a pancreatic β cell. In somecases, the genetically modified cell is a spleen, liver, peripheralblood, lymph nodes, thymus, or bone marrow cell. In some cases, thegenetically modified cell is a porcine cell. In some cases, thegenetically modified cell is from an embryotic tissue, a non-human fetalanimal, perinatal non-human animal, neonatal non-human animal,preweaning non-human animal, young adult non-human animal, or adultnon-human animal.

In another aspect, also disclosed herein is vaccine suitable for use ingenerating tolerance in a subject to transplanting a cell, tissue ororgan which comprises an injectable composition comprising cells asdefined in the application. Disclosed herein also includes a tolerizingvaccine comprising the genetically modified cell described in theapplication. In some cases, the genetically modified cell is anapoptotic cell. In some cases, the genetically modified cell is a fixedcell. In some cases, the vaccine further comprises a non-fixed cell. Insome cases, the fixed cell and the non-fixed cell are geneticallyidentical. In some cases, the fixed cell is fixed by a chemical and/orthe fixed cell induces anergy of immune cells in the subject. In somecases, the genetically modified cell is an1-Ethyl-3-(3-imethylaminopropyl)carbodiimide (ECDI)-fixed cell.

In another aspect, disclosed herein is a tissue or organ comprising thegenetically modified cell described in the application.

In another aspect, disclosed herein is a pancreas or pancreatic isletcomprising the genetically modified cell described herein.

In another aspect, disclosed herein is a pharmaceutical compositioncomprising the genetically modified cell described herein and apharmaceutically acceptable excipient.

In another aspect, disclosed herein is a genetically modified cell,tissue, or organ comprising a genetically modified cell for use intransplanting to a subject in need thereof to treat a condition in thesubject, where the subject is tolerized to the genetically modifiedcell, tissue, or organ by use of a vaccine. In some cases, the subjectis administered one or more pharmaceutical agents that inhibit T cellactivation, B cell activation, and/or dendritic cell activation.

In another aspect, disclosed herein is a method for treating a conditionin a subject in need thereof comprising a) transplanting the geneticallymodified cell, tissue or organ described in the application; b)administering a vaccine described in the application to the subject;and/or c) administering one or more pharmaceutical agents that inhibit Tcell activation, B cell activation, and/or dendritic cell activation tothe subject.

In another aspect, disclosed herein is a method for treating a conditionin a subject in need thereof comprising: a) administering a vaccine tothe subject; and b) transplanting a genetically modified cell, tissue,or organ comprising a genetically modified cell to the subject. In somecases, administering to the subject one or more pharmaceutical agentsthat inhibits T cell activation, B cell activation, and/or dendriticcell activation. In some cases, the transplanted genetically modifiedcell is the genetically modified cell described in the application. Insome cases, the vaccine is the vaccine described in the application. Insome cases, the vaccine comprises from or from about 0.001 to 1.0endotoxin unit per kg bodyweight of the subject. In some cases, thevaccine comprises from or from about 1 to 10 aggregates per μl. In somecases, the vaccine is administered 7 days before the transplantation and1 day after the transplantation. In some cases, the vaccine comprises atleast from or from about 1×10⁸ to 4×10⁸ splenocytes or splenic B cellsper kg bodyweight of the subject. In some cases, the splenocytes orsplenic B cells comprise from or from about 80% to 100% CD21 positiveSLA Class II positive B cells. In some cases, the vaccine is providedintravenously. In some cases, the transplanted cell, tissue, or organ isfunctional for at least 7 days after transplanted to the subject. Insome cases, the transplanting is xenotransplanting. In some cases, thepharmaceutical agent comprises a first dose of an anti-CD40 antibody. Insome cases, the first dose is given to the subject about 8 days beforethe transplantation. In some cases, the first dose comprises from orfrom about 30 mg to 70 mg of anti-CD40 antibody per kg body weight ofthe subject. In some cases, the method further comprises administeringone or more additional immunosuppression agents to the subject. In somecases, the one or more additional immunosuppression agents comprise aB-cell depleting antibody, an mTOR inhibitor, a TNF-alpha inhibitor, anIL-6 inhibitor, a complement C3 or C5 inhibitor, and/or a nitrogenmustard alkylating agent. In some cases, one of the additionalimmunosuppression agents is a nitrogen mustard alkylating agent. In somecases, one of the nitrogen mustard alkylating agent is cyclophosphamide.In some cases, the cyclophosphamide is administered 2 or 3 days afterthe administration of the vaccine.

In some cases, where the cyclophosphamide is administered at a dose offrom or from about 50 mg/kg/day and 60 mg/kg/day. In some cases, thesubject is a human subject. In some cases, the subject is a non-humananimal. In some cases, the non-human animal is a cat or a dog. In somecases, the condition is a disease. In some cases, the disease isdiabetes. In some cases, the diabetes is type 1 diabetes, type 2diabetes, surgical diabetes, cystic fibrosis-related diabetes, and/ormitochondrial diabetes.

In another aspect, disclosed herein is a method for immunotolerizing arecipient to a graft comprising providing to the recipient the vaccinedescribed in the application.

In another aspect, disclosed herein is method for treating a conditionin a subject in need thereof comprising transplanting the geneticallymodified cell described in the application.

In another aspect, disclosed herein is a genetically modified celldescribed in the application, or a tissue or organ comprising thegenetically modified cell, for use in transplanting to a subject in needthereof to treat a condition in the subject, where the subject istolerized to the genetically modified cell, tissue, or organ by thevaccine described in the application, and where one or morepharmaceutical agents that inhibit T cell activation, B cell activation,and/or dendritic cell activation, is administered to the subject. Insome cases the transplanting is xenotransplanting.

In another aspect, disclosed herein is a genetically modified celldescribed in the application, or a tissue or organ comprising thegenetically modified cell, for use in administering to a subject in needthereof to treat a condition in the subject.

In another aspect, disclosed herein is a vaccine described in theapplication for use in immunotolerizing a recipient to a graft.

In another aspect, disclosed herein is a method for making a geneticallymodified animal described in the application, comprising: a) obtaining acell with reduced expression of one or more of a component of a MHCI-specific enhanceosome, a transporter of a MHC I-binding peptide,and/or C3; b) generating an embryo from the cell; and c) growing theembryo into the genetically modified animal. In some cases, the cell isa zygote.

In another aspect, disclosed herein is a method for making a geneticallymodified animal described in the application, comprising: a) obtaining afirst cell with reduced expression of one or more of a component of aMHC I-specific enhanceosome, a transporter of a MHC I-binding peptide,and/or C3; b) transferring a nucleus of the first cell to a second cellto generate an embryo; and c) growing the embryo to the geneticallymodified animal. In some cases, the reducing is performed by geneediting. In some cases, the gene editing is performed using a CRISPR/cassystem.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 demonstrates an immunotherapeutic strategy centered around theuse of genetically modified cell and organ grafts lacking functionalexpression of MHC class I. The need for maintenance immunosuppressionrequired for the prevention of graft rejection is progressively reduced(or the applicability of transplantation of cell and organ xenograftsand the transplantation of stem cell-derived cellular allografts andxenografts is progressively increased) when the transplantation ofgenetically modified cells and organs is combined with transient use ofantagonistic anti-CD40 antibodies and even more when combined with theadministration of tolerizing vaccines comprising apoptotic donor cellsunder the cover of anti-CD40 antibodies.

FIG. 2 demonstrates one strategy of making genetically modified pigislet cells and tolerizing vaccines. Two clonal populations of pigs arecreated. One population having at least GGTA1 knocked out can be used tocreate a tolerizing vaccine. The other clonal population of pigs thathave at least GGTA1 and MHC I genes (e.g., NRLC5) knocked out, can beused for cell, tissues, and/or organ donors.

FIG. 3 demonstrates use of positive and tolerizing vaccines (alsoreferred to as a negative vaccine).

FIG. 4 demonstrates an exemplary approach to extending the survival ofxenografts in a subject with infusion of apoptotic donor splenocytes fortolerizing vaccination under the cover of transient immunosuppression.

FIG. 5 shows an exemplary approach to preventing rejection or extendingsurvival of xenografts in a recipient in the absence of chronic andgeneralized immunosuppression of the xenograft recipient. This exemplaryapproach includes and integrates three components: i) geneticallyengineered islets with deficient and/or reduced expression of αGal, MHCclass I, complement C3, and CXCL10 and transgenic expression the HLA-G;ii) genetically engineered donor apoptotic and non-apoptotic mononuclearcells (e.g., splenocytes) with deficient and/or reduced expression ofαGal, Neu5Gc, and Sda/CAD as well as transgenic expression of HLA-G withor without human CD47, human PD-L1, human PD-L2 (e.g., the geneticallyengineered vaccine); and iii) the administration of transientimmunosuppression including antagonistic anti-CD40 mAb, anti-CD20 mAb,rapamycin, and transient anti-inflammatory therapy including compstatin(e.g., the compstatin derivative APL-2), anti-IL-6 receptor mAb, andsoluble TNF receptor.

FIG. 6 demonstrates an exemplary protocol for transplant rejectionprophylaxis in a pig-to-cynomolgus monkey islet xenotransplantation. IE:islet equivalent; sTNFR: soluble TNF receptor (e.g., etanercept);α-IL-6R: anti-interleukin 6 receptor; Tx′d: transplanted.

FIGS. 7A-7E demonstrate a strategy for cloning a px330-Ga12-1 plasmidtargeting GGTA1. FIG. 7A shows a cloning strategy and oligonucleotides(SEQ ID NOs: 199-200, respectively, in order of appearance) for making aguide RNA targeting GGTA1. FIG. 7B shows an insertion site on the px330plasmid (SEQ ID NO: 201). FIG. 7C shows a flow chart demonstrating thecloning and verification strategy. FIG. 7D shows a cloning site (SEQ IDNO: 203) and sequencing primers (SEQ ID NOs: 202 and 204, respectively,in order of appearance). FIG. 7E shows sequencing results (SEQ ID NOs:205-207, respectively, in order of appearance).

FIGS. 8A-8E demonstrate a strategy for cloning a px330-CM1F plasmidtargeting CMAH. FIG. 8A shows a cloning strategy and oligonucleotides(SEQ ID NOs: 208-209, respectively, in order of appearance) for making aguide RNA targeting CMAH1. FIG. 8B shows an insertion site on the px330plasmid (SEQ ID NO: 210). FIG. 8C shows a flow chart demonstrating thecloning and verification strategy. FIG. 8D shows a cloning site (SEQ IDNO: 212) and sequencing primers (SEQ ID NOs: 211 and 213, respectively,in order of appearance). FIG. 8E shows sequencing results (SEQ ID NOs:214-216, respectively, in order of appearance).

FIGS. 9A-9E demonstrate a strategy for cloning a px330-NL1_FIRST plasmidtargeting NLRC5. FIG. 9A shows a cloning strategy and oligonucleotides(SEQ ID NOs: 217-218, respectively, in order of appearance) for making aguide RNA targeting NLRC5. FIG. 9B shows an insertion site on the px330plasmid (SEQ ID NO: 219). FIG. 9C shows a flow chart demonstrating thecloning and verification strategy. FIG. 9D shows a cloning site (SEQ IDNO: 221) and sequencing primers (SEQ ID NOs: 220 and 222, respectively,in order of appearance). FIG. 9E shows sequencing results (SEQ ID NOs:223-225, respectively, in order of appearance).

FIGS. 10A-10E demonstrate a strategy for cloning a px330/C3-5 plasmidtargeting C3. FIG. 10A shows a cloning strategy and oligonucleotides(SEQ ID NOs: 226-227, respectively, in order of appearance) for making aguide RNA targeting C3. FIG. 10B shows an insertion site on the px330plasmid (SEQ ID NO: 228). FIG. 10C shows a flow chart demonstrating thecloning and verification strategy. FIG. 10D shows a cloning site (SEQ IDNO: 230) and sequencing primers (SEQ ID NOs: 229 and 231, respectively,in order of appearance). FIG. 10E shows sequencing results (SEQ ID NOs:232-234, respectively, in order of appearance).

FIGS. 11A-11E demonstrate a strategy for cloning a px330/B41_secondplasmid targeting B4GALNT2. FIG. 11A shows a cloning strategy andoligonucleotides (SEQ ID NOs: 235-236, respectively, in order ofappearance) for making a guide RNA targeting B4GALNT2. FIG. 11B shows aninsertion site on the px330 plasmid (SEQ ID NO: 237). FIG. 11C shows aflow chart demonstrating the cloning and verification strategy. FIG. 11Dshows a cloning site (SEQ ID NO: 239) and sequencing primers (SEQ IDNOs: 238 and 240, respectively, in order of appearance). FIG. 11E showssequencing results (SEQ ID NOs: 241-243, respectively, in order ofappearance).

FIG. 12 demonstrates a map of Rosa26 locus sequenced in Example 2.

FIGS. 13A-13E demonstrate a strategy for cloning a px330/Rosa exon 1plasmid targeting Rosa26. FIG. 13A shows a cloning strategy andoligonucleotides (SEQ ID NOs: 244-245, respectively, in order ofappearance) for making a guide RNA targeting Rosa26. FIG. 13B shows aninsertion site on the px330 plasmid (SEQ ID NO: 246). FIG. 13C shows aflow chart demonstrating the cloning and verification strategy. FIG. 13Dshows a cloning site (SEQ ID NO: 248) and sequencing primers (SEQ IDNOs: 247 and 249, respectively, in order of appearance). FIG. 13E showssequencing results (SEQ ID NOs: 250-252, respectively, in order ofappearance).

FIG. 14A shows a map of the genomic sequence of GGTA1. FIG. 14B shows amap of the cDNA sequence of GGTA1.

FIG. 15 shows an exemplary microscopic view of porcine fetal fibroblaststransfected with pSpCas9(BB)-2A-GFP.

FIG. 16 shows a fluorescence in situ hybridization (FISH) to the GGTA1gene by specific probes revealing the location on chromosome 1.

FIGS. 17A-17B demonstrate an example of phenotypic selection of cellswith cas9/sgRNA-mediated GGTA1/NLCR5 disruption. FIG. 17A showsgenetically modified cells, which do not express alpha-galactosidase.FIG. 17B shows non-genetically modified cells, which expressalpha-galactosidase and were labeled with isolectin B4 (IB)-linkedferrous beads.

FIGS. 18A-18C demonstrates validation of GGTA1, CMAH, and NLRC5disruption in pig cells. FIG. 18A demonstrates validation of GGTA1disruption in pig cells. FIG. 18A discloses SEQ ID NOs: 253-255,respectively, in order of appearance. FIG. 18B demonstrates validationof CMAH disruption in pig cells. FIG. 18B discloses SEQ ID NOs: 256-258,respectively, in order of appearance. FIG. 18C demonstrates validationof NLRC5 disruption in pig cells. FIG. 18C discloses SEQ ID NOs:259-261, respectively, in order of appearance.

FIGS. 19A-19B demonstrate the inhibitory effects of an anti-SLA antibodyon the pig islet-induced human CD8+ T cell activation. FIG. 19A showsthe proliferation of CD8+ T cells, CD4 T cells and natural killer (NK)cells in a mixed culture with adult pig islets for 7 days in thepresence (black bars) or absence (white bars) of the anti-SLA antibody.FIG. 19B shows the viability (assessed by AO/PI staining) of adult pigislets cultured with or without highly purified lymphocytes for 7 daysin the presence (black bars) or absence (white bars) of the anti-SLAclass I antibody.

FIGS. 20A-20B demonstrate T cell activation induced by porcine islets.FIG. 20A demonstrates the result of ELISPOT assays. The results show thesuppression of a posttransplant increase of anti-donor T cells withdirect and indirect specificity secreting IFN-γ in a cynomolgus monkey.The monkey was treated with peritransplant infusion of apoptotic donorsplenocytes from a GT-KO donor pig, and islets from the same donor pigon day 0 under the cover of transient immunosuppression with anti-CD40monoclonal antibody, rapamycin, sTNFR, and anti-IL-6R monoclonalantibody. FIG. 20B demonstrates CD8 staining of an intraportallytransplanted adult porcine islet undergoing rejection at 141 days aftertransplantation.

FIGS. 21A-21D demonstrate porcine islet graft survival in a monkey inthe absence of maintenance immunosuppression. FIG. 21A demonstratesblood glucose levels and exogenous insulin needed to maintain normalblood glucose level before and after transplantation. FIG. 21Bdemonstrates serum porcine C-peptide level in a monkey. FIG. 21Cdemonstrates blood glucose levels in response to glucose challenges.FIG. 21D demonstrates serum porcine C-peptide levels in response toglucose challenges.

FIG. 22A demonstrates rejection of non-genetically modified porcineislets by a monkey transplanted with islets and receiving anti-CD40antibody four times through day 14 after transplantation and maintenanceimmunosuppression with CTLA4-Ig and rapamycin. FIG. 22B demonstratesamelioration of diabetes by transplanted porcine islets in monkeysreceiving anti-CD40 antibody four times through day 14 aftertransplantation and maintenance immunosuppression with CTLA4-Ig andrapamycin.

FIG. 23A demonstrates amelioration of diabetes (restoration of sustainednormoglycemia and insulin independence) by transplanted porcine isletsin a monkey (ID #13CP7) receiving maintenance immunosuppression withrapamycin and anti-CD40 antibody weekly after transplantation. Themonkey was given an anti-CD40 antibody and rapamycin for 21 daysstarting from the day of transplantation. FIG. 23B demonstrates serumporcine C-peptide levels (fasted, random, and stimulated) in the samerecipient (ID #13CP7).

FIG. 24 shows the increase of circulating CD8+CD2hi CD28− effectormemory T cells in two cynomolgus monkeys at the time of sacrifice (afterpresumed rejection) compared with baseline and the high prevalence ofCD8+CD2hi CD28− effector memory T cells within the CD8+ T cellcompartment in liver mononuclear cells at the time of sacrifice. Bothmonkeys received intraportal xenotransplants of adult porcine islets.Pre Tx: pretransplant; PBL: peripheral blood leukocyte; Sac: sacrifice;Lym: lymphocyte; LMNC: liver mononuclear cell.

FIG. 25 shows suppression of circulating CD8+CD2hi CD28− effector memoryT cells by peritransplant infusion of apoptotic donor splenocytes(MX-ECDI-vaccine) compared with controls that received the sametransient immunosuppression but no apoptotic donor splenocytes(MX-ECDI-controls). Pre Tx: pretransplant; Sac: sacrifice; Lym:lymphocyte; LMNC: liver mononuclear cell.

FIG. 26 shows suppression of circulating CD8+CD2hi CD28− effector memoryT cells by apoptotic donor splenocytes and α-CD40 antibodies. CM:cynomolgus monkey; Pre Tx: pretransplant; D: day.

FIG. 27 shows suppression of circulating CD4+CD25hi FoxP3+CD127lowregulatory T cells by apoptotic donor splenocytes and α-CD40 antibodies.CM: cynomolgus monkey; Pre Tx: pretransplant; D: day.

FIG. 28 shows suppression of circulating CD8+CD122+ natural suppressorcells by apoptotic donor splenocytes and α-CD40 antibodies. CM:cynomolgus monkey; Pre Tx: pretransplant; D: day.

FIGS. 29A-29B show sequencing of DNA isolated from fetal cells of twoseparate litters (Pregnancy 1: FIG. 29A or Pregnancy 2: FIG. 29B)subjected to PCR amplification of the GGTA1 (compared to Sus scrofabreed mixed chromosome 1, Sscrofa10.2 NCBI Reference Sequence:NC_010443.4) target regions and the resulting amplicons were separatedon 1% agarose gels. Amplicons were also analyzed by sanger sequencingusing the forward primer alone from each reaction. In FIG. 29A, theresults are shown from Pregnancy 1's fetuses 1, 2, 4, 5, 6, and 7,truncated 6 nucleotides after the target site for GGTA1. Fetus 3 wastruncated 17 nucleotides after the cut site followed by a 2,511(668-3179) nucleotide deletion followed by a single base substitution.Truncation, deletion and substitution from a single sequencingexperiment containing the alleles from both copies of the target genecan only suggest a gene modification has occurred but not reveal theexact sequence for each allele. From this analysis it appears that all 7fetuses have a single allele modification for GGTA1. FIG. 29A disclosesSEQ ID NOs: 262-271, respectively, in order of appearance. FIG. 29Bshows pregnancy 2 fetal DNA samples 1, 3, 4, and 5 were truncated 3nucleotides from the GGTA1 gene target site. Fetus 2 had variability insanger sequencing that suggests a complex variability in DNA mutationsor poor sample quality. However, fetal DNA template quality wassufficient for the generation of the GGTA1 gene screening experimentdescribed above. FIG. 29B discloses SEQ ID NOs: 272-278, respectively,in order of appearance.

FIGS. 30A-30B show sequencing of DNA isolated from fetal cells of twoseparate litters (Pregnancy 1: FIG. 30A or Pregnancy 2: FIG. 30B)subjected to PCR amplification of the NLRC5 (consensus sequence) targetregions and the resulting amplicons were separated on 1% agarose gels.Amplicons were also analyzed by sanger sequencing using the forwardprimer alone from each reaction. Sequence analysis of the NLRC5 targetsite for fetuses from Pregnancy 1 (FIG. 30A) was unable to showconsistent alignment suggesting an unknown complication in thesequencing reaction or varying DNA modifications between NLRC5 allelesthat complicate the sanger sequencing reaction and analysis. FIG. 30Adiscloses SEQ ID NOs: 279-294, respectively, in order of appearance.NLRC5 gene amplicons from Pregnancy 2 (FIG. 30B) were all truncated 120nucleotides downstream of the NLRC5 gene cut site. FIG. 30B disclosesSEQ ID NOs: 295-310, respectively, in order of appearance.

FIGS. 31A-31B show data from fetal DNA (wt and 1-7 (FIG. 31A:Pregnancy 1) or 1-5 (FIG. 31B: Pregnancy 2) isolated from hind limbbiopsies. Target genes were amplified by PCR and PCR products wereseparated on 1% agarose gels and visualized by fluorescent DNA stain.The amplicon band present in the wt lanes represent the unmodified DNAsequence. An increase or decrease in size of the amplicon suggests aninsertion or deletion within the amplicon, respectively. Variation inthe DNA modification between alleles in one sample may make the bandappear more diffuse. Pregnancy 1 (FIG. 31A) resulted in 7 fetuses whilepregnancy 2 (FIG. 31B) resulted in 5 fetuses harvested at 45 and 43days, respectively. A lack of band as in the NLRC5 gel in fetuses 1, 3,and 4 of FIG. 31A (bottom gel), suggests that the modification to thetarget region have disrupted the binding of DNA amplification primers.The presence of all bands in GGTA 1 in FIG. 31A (top gel) suggests thatDNA quality was sufficient to generate DNA amplicons in the NLRC5targeting PCR reactions. Fetuses 1, 2, 4, and 5 of Pregnancy 1 (FIG.31A) have larger GGTA 1 amplicons than the WT suggesting an insertionwithin the target area. In fetus 3 of Pregnancy 1 (FIG. 31A), the GGTA 1amplicon migrated faster than the WT control suggesting a deletionwithin the target area. Fetuses 6 and 7 of Pregnancy 1 (FIG. 31A) NLRC5amplicons migrated faster than the WT suggesting a deletion within thetarget area. Fetuses 1-5 (FIG. 31B) GGTA1 amplicons were difficult tointerpret by size and were diffuse as compared to the WT control.Fetuses 1-5 (FIG. 31B) NLRC5 amplicons were uniform in size and densityas compared to the wild type control.

FIGS. 32A-32E shows phenotypic analysis of fetuses from two separatelitters of pigs (FIGS. 32A-32C: Pregnancy 1 or FIGS. 32D-32E: Pregnancy2). Fetuses were harvested at day 45 (Pregnancy 1) or 43 days (Pregnancy2) and processed for DNA and culture cell isolation. Tissue fragmentsand cells were plated in culture media for 2 days to allow fetal cellsto adhere and grow. Wild type cells (fetal cells not geneticallymodified) and fetal cells from pregnancy 1 and 2 were removed fromculture plates and labeled with IB4 lectin conjugated to alexa fluor 488or anti-porcine MHC class I antibody conjugated to FITC. Flow cytometricanalysis is shown as histograms depicting the labeling intensity of thecells tested. The histogram for the WT cells are included in each panelto highlight the decrease in overall intensity of each group of fetalcells. There is a decrease in alpha Gal and MHC class I labeling inpregnancy 1 (FIGS. 32A-32C) indicated as a decrease in peak intensity.In pregnancy 2 (FIGS. 32D-32E) fetuses 1 and 3 have a large decrease inalpha gal labeling and significant reduction in MHC class 1 labeling ascompared to WT fetal cells.

FIGS. 33A-33C shows the impact of decreased MHC class I expression incells from Fetus 3 (Pregnancy 1) as compared to wild type fetal cellsfrom a genetic clone. The proliferative response of human CD8+ cells andCD4 T cells to porcine control fibroblast and NLRC5 knockout fetal cellswere measured. FIG. 33A. Cells were gated as CD4 or CD8 beforeassessment of proliferation. FIG. 33B. CD8 T cell proliferation wasreduced following treatments stimulation by porcine fetal GGTA1/NLRC5knockout cells compared to control unmodified porcine fibroblast. Almosta 55% reduction in CD8 T cells proliferation was observed when humanresponders were treated with porcine fetal GGTA1/NLRC5 knockout cells at1:1 ratio. Wild type fetal cells elicited a 17.2% proliferation in humanCD8 T cells whereas the MHC class I deficient cells from fetus 3(Pregnancy 1) induced only a 7.6% proliferation. No differences wereseen in CD8 T cells proliferative response at 1:5 and 1:10 ratiocompared to unmodified fetal cells. FIG. 33C. No changes were observedin CD4 T cell proliferation in response to NLRC5 knockout and controlunmodified porcine fetal cells at all ratios studied.

DETAILED DESCRIPTION OF THE DISCLOSURE

The following description and examples illustrate embodiments of theinvention in detail. It is to be understood that this invention is notlimited to the particular embodiments described herein and as such canvary. Those of skill in the art will recognize that there are numerousvariations and modifications of this invention, which are encompassedwithin its scope.

Failure of organ and tissue function can result in premature death ofindividuals. Transplantation can potentially solve this problem, whichcan prolong the lives of many individuals. However, there is a shortageof cells, organs, and/or tissues that can be used for transplantation.

Xenografts or allografts (e.g., embryonic or induced pluripotent stemcells) can be used to create an unlimited supply of cells, organs,and/or tissues used for transplantation. In general, sometransplantation can lead to increased immune response which canultimately lead to transplantation rejection. Isografts or autograftstypically do not result in rejection. However, allografts and xenograftscan result in immune reaction and can ultimately lead to the destructionof the graft. The risk of rejection in some cases can be mitigated bysuppressing the immune response.

Traditionally, immunosuppressive drugs were used after transplantation.However, there are many detrimental effects associated with long-termtreatment with immunosuppressive drugs, including but not limited toincreased risk of cancer and infection. Alternative methods to preventgraft rejection and suppress the immune system were sought. The immuneresponse can be tempered by use of various techniques, including thosedescribed herein. For example, one method described herein to preventtransplantation rejection or prolong the time to transplantationrejection without or with minimal immunosuppressive drug use, an animal,e.g., a donor non-human animal, could be altered, e.g., genetically.Subsequently, the cells, organs, and/or tissues of the altered animal,e.g., a donor non-human animal, can be harvested and used in allograftsor xenografts. Alternatively, cells can be extracted from an animal,e.g., a human or non-human animal (including but not limited to primarycells) or cells can be previously extracted animal cells, e.g., celllines. These cells can be used to create a genetically altered cell.

Transplant rejection (e.g., T cells-mediated transplant rejection) canbe prevented by chronic immunosuppression. However, immunosuppression iscostly and associated with the risk of serious side effects. Tocircumvent the need for chronic immunosuppression, a multifaceted, Tcell-targeted rejection prophylaxis was developed (FIG. 1) that

-   -   i) utilizes genetically modified grafts lacking functional        expression of MHC class I, thereby interfering with activation        of CD8⁺ T cells with direct specificity and precluding cytolytic        effector functions of these CD8⁺ T cells,    -   ii) interferes with B cell (and other APC)-mediated priming and        memory generation of anti-donor T cells using induction        immunotherapy comprising antagonistic anti-CD40 mAbs (and        depleting anti-CD20 mAbs and a mTOR inhibitor), and/or    -   iii) deletes anti-donor T cells with indirect specificity via        peritransplant infusions of apoptotic donor cell vaccines.

Described herein are genetically modified non-human animals (such asnon-human primates or a genetically modified animal that is member ofthe Laurasiatheria superorder, e.g., ungulates) and organs, tissues, orcells isolated therefrom, tolerizing vaccines, and methods for treatingor preventing a disease in a recipient in need thereof bytransplantation of an organ, tissue, or cell isolated from a non-humananimal. An organ, tissue, or cell isolated from a non-human animal (suchas non-human primates or a genetically modified animal that is member ofthe Laurasiatheria superorder, e.g., ungulates) can be transplanted intoa recipient in need thereof from the same species (an allotransplant) ora different species (a xenotransplant). A recipient can be tolerizedwith a tolerizing vaccine and/or one or more immunomodulatory agents(e.g., an antibody). In embodiments involving xenotransplantation therecipient can be a human. Suitable diseases that can be treated are anyin which an organ, tissue, or cell of a recipient is defective orinjured, (e.g., a heart, lung, liver, vein, skin, or pancreatic isletcell) and a recipient can be treated by transplantation of an organ,tissue, or cell isolated from a non-human animal.

Definitions

The term “about” in relation to a reference numerical value and itsgrammatical equivalents as used herein can include the numerical valueitself and a range of values plus or minus 10% from that numericalvalue. For example, the amount “about 10” includes 10 and any amountsfrom 9 to 11. For example, the term “about” in relation to a referencenumerical value can also include a range of values plus or minus 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

The term “non-human animal” and its grammatical equivalents as usedherein includes all animal species other than humans, includingnon-human mammals, which can be a native animal or a geneticallymodified non-human animal. A non-human mammal includes, an ungulate,such as an even-toed ungulate (e.g., pigs, peccaries, hippopotamuses,camels, llamas, chevrotains (mouse deer), deer, giraffes, pronghorn,antelopes, goat-antelopes (which include sheep, goats and others), orcattle) or an odd-toed ungulate (e.g., horse, tapirs, and rhinoceroses),a non-human primate (e.g., a monkey, or a chimpanzee), a Canidae (e.g.,a dog) or a cat. A non-human animal can be a member of theLaurasiatheria superorder. The Laurasiatheria superorder can include agroup of mammals as described in Waddell et al., Towards Resolving theInterordinal Relationships of Placental Mammals. Systematic Biology 48(1): 1-5 (1999). Members of the Laurasiatheria superorder can includeEulipotyphla (hedgehogs, shrews, and moles), Perissodactyla(rhinoceroses, horses, and tapirs), Carnivora (carnivores),Cetartiodactyla (artiodactyls and cetaceans), Chiroptera (bats), andPholidota (pangolins). A member of Laurasiatheria superorder can be anungulate described herein, e.g., an odd-toed ungulate or even-toedungulate. An ungulate can be a pig. A member can be a member ofCarnivora, such as a cat, or a dog. In some cases, a member of theLaurasiatheria superorder can be a pig.

The term “pig” and its grammatical equivalents as used herein can referto an animal in the genus Sus, within the Suidae family of even-toedungulates. For example, a pig can be a wild pig, a domestic pig, minipigs, a Sus scrofa pig, a Sus scrofa domesticus pig, or inbred pigs.

The term “transgene” and its grammatical equivalents as used herein canrefer to a gene or genetic material that can be transferred into anorganism. For example, a transgene can be a stretch or segment of DNAcontaining a gene that is introduced into an organism. When a transgeneis transferred into an organism, the organism can then be referred to asa transgenic organism. A transgene can retain its ability to produce RNAor polypeptides (e.g., proteins) in a transgenic organism. A transgenecan comprise a polynucleotide encoding a protein or a fragment (e.g., afunctional fragment) thereof. The polynucleotide of a transgene can bean exogenous polynucleotide. A fragment (e.g., a functional fragment) ofa protein can comprise at least or at least about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amino acid sequence ofthe protein. A fragment of a protein can be a functional fragment of theprotein. A functional fragment of a protein can retain part or all ofthe function of the protein.

The term “genetic modification” and its grammatical equivalents as usedherein can refer to one or more alterations of a nucleic acid, e.g., thenucleic acid within an organism's genome. For example, geneticmodification can refer to alterations, additions, and/or deletion ofgenes. A genetically modified cell can also refer to a cell with anadded, deleted and/or altered gene. A genetically modified cell can befrom a genetically modified non-human animal. A genetically modifiedcell from a genetically modified non-human animal can be a cell isolatedfrom such genetically modified non-human animal. A genetically modifiedcell from a genetically modified non-human animal can be a celloriginated from such genetically modified non-human animal. For example,a cell

The term “islet” or “islet cells” and their grammatical equivalents asused herein can refer to endocrine (e.g., hormone-producing) cellspresent in the pancreas of an organism. For example, islet cells cancomprise different types of cells, including, but not limited to,pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic Fcells, and/or pancreatic ε cells. Islet cells can also refer to a groupof cells, cell clusters, or the like.

The term “condition” condition and its grammatical equivalents as usedherein can refer to a disease, event, or change in health status.

The term “diabetes” and its grammatical equivalents as used herein canrefer to is a disease characterized by high blood sugar levels over aprolonged period. For example, the term “diabetes” and its grammaticalequivalents as used herein can refer to all or any type of diabetes,including, but not limited to, type 1, type 2, cystic fibrosis-related,surgical, gestational diabetes, and mitochondrial diabetes. In somecases, diabetes can be a form of hereditary diabetes.

The term “phenotype” and its grammatical equivalents as used herein canrefer to a composite of an organism's observable characteristics ortraits, such as its morphology, development, biochemical orphysiological properties, phenology, behavior, and products of behavior.Depending on the context, the term “phenotype” can sometimes refer to acomposite of a population's observable characteristics or traits.

The term “disrupting” and its grammatical equivalents as used herein canrefer to a process of altering a gene, e.g., by deletion, insertion,mutation, rearrangement, or any combination thereof. For example, a genecan be disrupted by knockout. Disrupting a gene can be partiallyreducing or completely suppressing expression (e.g., mRNA and/or proteinexpression) of the gene. Disrupting can also include inhibitorytechnology, such as shRNA, siRNA, microRNA, dominant negative, or anyother means to inhibit functionality or expression of a gene or protein.

The term “gene editing” and its grammatical equivalents as used hereincan refer to genetic engineering in which one or more nucleotides areinserted, replaced, or removed from a genome. For example, gene editingcan be performed using a nuclease (e.g., a natural-existing nuclease oran artificially engineered nuclease).

The term “transplant rejection” and its grammatical equivalents as usedherein can refer to a process or processes by which an immune responseof an organ transplant recipient mounts a reaction against thetransplanted material (e.g., cells, tissues, and/or organs) sufficientto impair or destroy the function of the transplanted material.

The term “hyperacute rejection” and its grammatical equivalents as usedherein can refer to rejection of a transplanted material or tissueoccurring or beginning within the first 24 hours after transplantation.For example, hyperacute rejection can encompass but is not limited to“acute humoral rejection” and “antibody-mediated rejection”.

The term “negative vaccine”, “tolerizing vaccine” and their grammaticalequivalents as used herein, can be used interchangeably. A tolerizingvaccine can tolerize a recipient to a graft or contribute totolerization of the recipient to the graft if used under the cover ofappropriate immunotherapy. This can help to prevent transplantationrejection.

The term “recipient”, “subject” and their grammatical equivalents asused herein, can be used interchangeably. A recipient or a subject canbe a human or non-human animal. A recipient or a subject can be a humanor non-human animal that will receive, is receiving, or has received atransplant graft, a tolerizing vaccine, and/or other compositiondisclosed in the application. A recipient or subject can also be in needof a transplant graft, a tolerizing vaccine and/or other compositiondisclosed in the application. In some cases, a recipient can be a humanor non-human animal that will receive, is receiving, or has received atransplant graft.

Some numerical values disclosed throughout are referred to as, forexample, “X is at least or at least about 100; or 200 [or any numericalnumber].” This numerical value includes the number itself and all of thefollowing:

i) X is at least 100;

ii) X is at least 200;

iii) X is at least about 100; and

iv) X is at least about 200.

All these different combinations are contemplated by the numericalvalues disclosed throughout. All disclosed numerical values should beinterpreted in this manner, whether it refers to an administration of atherapeutic agent or referring to days, months, years, weight, dosageamounts, etc., unless otherwise specifically indicated to the contrary.

The ranges disclosed throughout are sometimes referred to as, forexample, “X is administered on or on about day 1 to 2; or 2 to 3 [or anynumerical range].” This range includes the numbers themselves (e.g., theendpoints of the range) and all of the following:

i) X being administered on between day 1 and day 2;

ii) X being administered on between day 2 and day 3;

iii) X being administered on between about day 1 and day 2;

iv) X being administered on between about day 2 and day 3;

v) X being administered on between day 1 and about day 2;

vi) X being administered on between day 2 and about day 3;

vii) X being administered on between about day 1 and about day 2; and

viii) X being administered on between about day 2 and about day 3.

All these different combinations are contemplated by the rangesdisclosed throughout. All disclosed ranges should be interpreted in thismanner, whether it refers to an administration of a therapeutic agent orreferring to days, months, years, weight, dosage amounts, etc., unlessotherwise specifically indicated to the contrary.

The terms “and/or” and “any combination thereof” and their grammaticalequivalents as used herein, can be used interchangeably. These terms canconvey that any combination is specifically contemplated. Solely forillustrative purposes, the following phrases “A, B, and/or C” or “A, B,C, or any combination thereof” can mean “A individually; B individually;C individually; A and B; B and C; A and C; and A, B, and C.”

I. Genetically Modified Non-Human Animals

Provided herein are genetically modified animals that can be donors ofcells, tissues, and/or organs for transplantation. A geneticallymodified non-human animal can be any desired species. For example, agenetically modified non-human animal described herein can be agenetically modified non-human mammal. A genetically modified non-humanmammal can be a genetically modified ungulate, including a geneticallymodified even-toed ungulate (e.g., pigs, peccaries, hippopotamuses,camels, llamas, chevrotains (mouse deer), deer, giraffes, pronghorn,antelopes, goat-antelopes (which include sheep, goats and others), orcattle) or a genetically modified odd-toed ungulate (e.g., horse,tapirs, and rhinoceroses), a genetically modified non-human primate(e.g., a monkey, or a chimpanzee) or a genetically modified Canidae(e.g., a dog). A genetically modified non-human animal can be a memberof the Laurasiatheria superorder. A genetically modified non-humananimal can be a non-human primate, e.g., a monkey, or a chimpanzee. If anon-human animal is a pig, the pig can be at least or at least about 5,50, 100, or 300 pounds, e.g., the pig can be or be about between 5pounds to 50 pounds; 25 pounds to 100 pounds; or 75 pounds to 300pounds. In some cases, a non-human animal is a pig that has given birthat least one time.

A genetically modified non-human animal can be of any age. For example,the genetically modified non-human animal can be a fetus; from or fromabout 1 day to 1 month; from or from about 1 month to 3 months; from orfrom about 3 months to 6 months; from or from about 6 months to 9months; from or from about 9 months to 1 year; from or from about 1 yearto 2 years. A genetically modified non-human animal can be a non-humanfetal animal, perinatal non-human animal, neonatal non-human animal,preweaning non-human animal, young adult non-human animal, or an adultnon-human animal.

A genetically modified non-human animal can comprise reduced expressionof one or more genes compared to a non-genetically modified counterpartanimal. A non-genetically modified counterpart animal can be an animalsubstantially identical to the genetically modified animal but withoutgenetic modification in the genome. For example, a non-geneticallymodified counterpart animal can be a wild-type animal of the samespecies as the genetically modified animal. The non-human animal canprovide cells, tissues or organs for transplanting to a recipient orsubject in need thereof. A recipient or subject in need thereof can be arecipient or subject known or suspected of having a condition. Thecondition can be treated, prevented, reduced, eliminated, or augmentedby the methods and compositions disclosed herein. The recipient canexhibit low or no immuno-response to the transplanted cells, tissues ororgans. The transplanted cells, tissues or organs can benon-recognizable by CD8+ T cells, NK cells, or CD4+ T cells of therecipient (e.g., a human or another animal). The genes whose expressionis reduced can include MHC molecules, regulators of MHC moleculeexpression, and genes differentially expressed between the donornon-human animal and the recipient (e.g., a human or another animal).The reduced expression can be mRNA expression or protein expression ofthe one or more genes. For example, the reduced expression can beprotein expression of the one or more genes. Reduced expression can alsoinclude no expression. For example an animal, cell, tissue or organ withreduced expression of a gene can have no expression (e.g., mRNA and/orprotein expression) of the gene. Reduction of expression of a gene caninactivate the function of the gene. In some cases, when expression of agene is reduced in a genetically modified animal, the expression of thegene is absent in the genetically modified animal.

The genetically modified non-human animal can comprise reducedexpression of one or more MHC molecules compared to a non-geneticallymodified counterpart animal. For example, the non-human animal can be anungulate, e.g., a pig, with reduced expression of one or more swineleukocyte antigen (SLA) class I and/or SLA class II molecules.

The genetically modified non-human animal can comprise reducedexpression of any genes that regulate major histocompatibility complex(MHC) molecules (e.g., MHC I molecules and/or MHC II molecules) comparedto a non-genetically modified counterpart animal. Reducing expression ofsuch genes can result in reduced expression and/or function of MHCmolecules (e.g., MHC I molecules and/or MHC II molecules). In somecases, the one or more genes whose expression is reduced in thenon-human animal can comprise one or more of the following: componentsof an MHC I-specific enhanceosome, transporters of a MHC I-bindingpeptide, natural killer group 2D ligands, CXC chemical receptor (CXCR) 3ligands, complement component 3 (C3), and major histocompatibilitycomplex II transactivator (CIITA). In some cases, the component of a MHCI-specific enhanceosome can be NLRC5. In some cases, the component of aMHC I-specific enhanceosome can also comprise regulatory factor X (RFX)(e.g., RFX1), nuclear transcription factor Y (NFY), and cAMP responseelement-binding protein (CREB). In some instances, the transporter of aMHC I-binding peptide can be Transporter associated with antigenprocessing 1 (TAP1). In some cases, the natural killer (NK) group 2Dligands can comprise MICA and MICB. For example, the geneticallymodified non-human animal can comprise reduced expression of one or moreof the following genes: NOD-like receptor family CARD domain containing5 (NLRC5), Transporter associated with antigen processing 1 (TAP1),C-X-C motif chemokine 10 (CXCL10), MHC class I polypeptide-relatedsequence A (MICA), MHC class I polypeptide-related sequence B (MICB),complement component 3 (C3), and CIITA. A genetically modified animalcan comprise reduced expression of one or more of the following genes: acomponent of an MHC I-specific enhanceosome (e.g., NLRC5), a transporterof an MHC I-binding peptide (TAP1), and C3.

The genetically modified non-human animal can comprise reducedexpression compared to a non-genetically modified counterpart of one ormore genes expressed at different levels between the non-human animaland a recipient receiving a cell, tissue, or organ from the non-humananimal. For example, the one or more genes can be expressed at a lowerlevel in a human than in the non-human animal. In some cases, the one ormore genes can be endogenous genes of the non-human animal. Theendogenous genes are in some cases genes not expressed in anotherspecies. For example, the endogenous genes of the non-human animal canbe genes that are not expressed in a human. For example, in some cases,homologs (e.g., orthologs) of the one or more genes do not exist in ahuman. In another example, homologs (e.g., orthologs) of the one or moregenes can exist in a human but are not expressed.

In some cases, the non-human animal can be a pig, and the recipient canbe a human. In these cases, the one or more genes can be any genesexpressed in a pig but not in a human. For example, the one or moregenes can comprise glycoprotein galactosyltransferase alpha 1,3 (GGTA1),putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-likeprotein (CMAH), and β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). Agenetically modified non-human animal can comprise reduced expression ofB4GALNT2, GGTA1, or CMAH, where the reduced expression is in comparisonto a non-genetically modified counterpart animal. A genetically modifiednon-human animal can comprise reduced expression of B4GALNT2 and GGTA1,where the reduced expression is in comparison to a non-geneticallymodified counterpart animal. A genetically modified non-human animal cancomprise reduced expression of B4GALNT2 and CMAH, where the reducedexpression is in comparison to a non-genetically modified counterpartanimal. A genetically modified non-human animal can comprise reducedexpression of B4GALNT2, GGTA1, and CMAH, where the reduced expression isin comparison to a non-genetically modified counterpart animal.

The genetically modified non-human animal can comprise reducedexpression compared to a non-genetically modified counterpart of one ormore of any of the genes disclosed herein, including NLRC5, TAP1,CXCL10, MICA, MICB, C3, CIITA, GGTA1, CMAH, and B4GALNT2.

A genetically modified non-human animal can comprise one or more geneswhose expression is reduced, e.g., where genetic expression is reduced.The one or more genes whose expression is reduced include but are notlimited to NOD-like receptor family CARD domain containing 5 (NLRC5),Transporter associated with antigen processing 1 (TAP1), Glycoproteingalactosyltransferase alpha 1,3 (GGTA1), Putative cytidinemonophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH),C-X-C motif chemokine 10 (CXCL10), MHC class I polypeptide-relatedsequence A (MICA), MHC class I polypeptide-related sequence B (MICB),class II major histocompatibility complex transactivator (CIITA),Beta-1,4-N-Acetyl-Galactosaminyl Transferase 2 (B4GALNT2), complementalcomponent 3 (C3), and/or any combination thereof.

A genetically modified non-human animal can comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more genes whoseexpression is disrupted. For illustrative purposes, and not to limitvarious combinations a person of skill in the art can envision, agenetically modified non-human animal can have NLRC5 and TAP1individually disrupted. A genetically modified non-human animal can alsohave both NLRC5 and TAP1 disrupted. A genetically modified non-humananimal can also have NLRC5 and TAP1, and in addition to one or more ofthe following GGTA1, CMAH, CXCL10, MICA, MICB, B4GALNT2, or CIITA genesdisrupted; for example “NLRC5, TAP1, and GGTA1” or “NLRC5, TAP1, andCMAH” can be disrupted. A genetically modified non-human animal can alsohave NLRC5, TAP1, GGTA1, and CMAH disrupted. Alternatively, agenetically modified non-human animal can also have NLRC5, TAP1, GGTA1,B4GALNT2, and CMAH disrupted. In some cases, a genetically modifiednon-human animal can have C3 and GGTA1 disrupted. In some cases, agenetically modified non-human animal can have reduced expression ofNLRC5, C3, GGTA1, B4GALNT2, CMAH, and CXCL10. In some cases, agenetically modified non-human animal can have reduced expression ofTAP1, C3, GGTA1, B4GALNT2, CMAH, and CXCL10. In some cases, agenetically modified non-human animal can have reduced expression ofNLRC5, TAP1, C3, GGTA1, B4GALNT2, CMAH, and CXCL10.

Lack of MHC class I expression on transplanted human cells can cause thepassive activation of natural killer (NK) cells (Ohlen et al., 1989).Lack of MHC class I expression could be due to NLRC5, TAP1, or B2M genedeletion. NK cell cytotoxicity can be overcome by the expression of thehuman MHC class 1 gene, HLA-E, can stimulate the inhibitory receptorCD94/NKG2A on NK cells to prevent cell killing (Weiss et al., 2009;Lilienfeld et al., 2007; Sasaki et al., 1999). Successful expression ofthe HLA-E gene can be dependent on co-expression of the human B2M (beta2 microglobulin) gene and a cognate peptide (Weiss et al., 2009;Lilienfeld et al., 2007; Sasaki et al., 1999; Pascasova et al., 1999). Anuclease mediated break in the stem cell DNA can allow for the insertionof one or multiple genes via homology directed repair. The HLA-E andhB2M genes in series can be integrated in the region of the nucleasemediated DNA break thus preventing expression of the target gene (forexample, NLRC5) while inserting the transgenes.

Expression levels of genes can be reduced to various extents. Forexample, expression of one or more genes can be reduced by or by about100%. In some cases, expression of one or more genes can be reduced byor by about 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% ofnormal expression, e.g., compared to the expression of non-modifiedcontrols. In some cases, expression of one or more genes can be reducedby at least or to at least about 99% to 90%; 89% to 80%, 79% to 70%; 69%to 60%; 59% to 50% of normal expression, e.g., compared to theexpression of non-modified controls. For example, expression of one ormore genes can be reduced by at least or at least about 90% or by atleast or at least about 90% to 99% of normal expression.

Expression can be measured by any known method, such as quantitative PCR(qPCR), including but not limited to PCR, real-time PCR (e.g.,Sybr-green), and/or hot PCR. In some cases, expression of one or moregenes can be measured by detecting the level of transcripts of thegenes. For example, expression of one or more genes can be measured byNorthern blotting, nuclease protection assays (e.g., RNase protectionassays), reverse transcription PCR, quantitative PCR (e.g., real-timePCR such as real-time quantitative reverse transcription PCR), in situhybridization (e.g., fluorescent in situ hybridization (FISH)), dot-blotanalysis, differential display, serial analysis of gene expression,subtractive hybridization, microarrays, nanostring, and/or sequencing(e.g., next-generation sequencing). In some cases, expression of one ormore genes can be measured by detecting the level of proteins encoded bythe genes. For example, expression of one or more genes can be measuredby protein immunostaining, protein immunoprecipitation, electrophoresis(e.g., SDS-PAGE), Western blotting, bicinchoninic acid assay,spectrophotometry, mass spectrometry, enzyme assays (e.g., enzyme-linkedimmunosorbent assays), immunohistochemistry, flow cytometry, and/orimmunoctyochemistry. Expression of one or more genes can also bemeasured by microscopy. The microscopy can be optical, electron, orscanning probe microscopy. Optical microscopy can comprise use of brightfield, oblique illumination, cross-polarized light, dispersion staining,dark field, phase contrast, differential interference contrast,interference reflection microscopy, fluorescence (e.g., when particles,e.g., cells, are immunostained), confocal, single plane illuminationmicroscopy, light sheet fluorescence microscopy, deconvolution, orserial time-encoded amplified microscopy. Expression of MHC I moleculescan also be detected by any methods for testing expression as describedherein.

Disrupted Genes

The inventors have found that cells, organs, and/or tissues havingdifferent combinations of disrupted genes, can result in cells, organs,and/or tissues that are less susceptible to rejection when transplantedinto a recipient. For example, the inventors have found that disrupting(e.g., reducing expression of) certain genes, such as NLRC5, TAP1,GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, C3, and/or CIITA, canincrease the likelihood of graft survival.

However, the disruptions are not limited to solely these genes. Thedisruption can be of any particular gene. It is contemplated thatgenetic homologues (e.g., any mammalian version of the gene) of thegenes within this applications are covered. For example, genes that aredisrupted can exhibit a certain identity and/or homology to genesdisclosed herein, e.g., NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10,MICA, MICB, C3, and/or CIITA. Therefore, it is contemplated that a genethat exhibits at least or at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99%, or 100% homology (at the nucleic acid orprotein level) can be disrupted, e.g., a gene that exhibits at least orat least about from 50% to 60%; 60% to 70%; 70% to 80%; 80% to 90%; or90% to 99% homology. It is also contemplated that a gene that exhibitsat least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,99%, or 100% identity (at the nucleic acid or protein level) can bedisrupted, e.g., a gene that exhibits at least or at least about from50% to 60%; 60% to 70%; 70% to 80%; 80% to 90%; or 90% to 99% identity.Some genetic homologues are known in the art, however, in some cases,homologues are unknown. However, homologous genes between mammals can befound by comparing nucleic acid (DNA or RNA) sequences or proteinsequences using publically available databases such as NCBI BLAST.Genomic sequences, cDNA and protein sequences of exemplary genes areshown in Table 1.

Gene suppression can also be done in a number of ways. For example, geneexpression can be reduced by knock out, altering a promoter of a gene,and/or by administering interfering RNAs (knockdown). This can be doneat an organism level or at a tissue, organ, and/or cellular level. Ifone or more genes are knocked down in a non-human animal, cell, tissue,and/or organ, the one or more genes can be reduced by administrating RNAinterfering reagents, e.g., siRNA, shRNA, or microRNA. For example, anucleic acid which can express shRNA can be stably transfected into acell to knockdown expression. Furthermore, a nucleic acid which canexpress shRNA can be inserted into the genome of a non-human animal,thus knocking down a gene with in a non-human animal.

Disruption methods can also comprise overexpressing a dominant negativeprotein. This method can result in overall decreased function of afunctional wild-type gene. Additionally, expressing a dominant negativegene can result in a phenotype that is similar to that of a knockoutand/or knockdown.

Sometimes a stop codon can be inserted or created (e.g., by nucleotidereplacement), in one or more genes, which can result in a nonfunctionaltranscript or protein (sometimes referred to as knockout). For example,if a stop codon is created within the middle of one or more genes, theresulting transcription and/or protein can be truncated, and can benonfunctional. However, in some cases, truncation can lead to an active(a partially or overly active) protein. In some cases, if a protein isoverly active, this can result in a dominant negative protein, e.g., amutant polypeptide that disrupts the activity of the wild-type protein.

This dominant negative protein can be expressed in a nucleic acid withinthe control of any promoter. For example, a promoter can be a ubiquitouspromoter. A promoter can also be an inducible promoter, tissue specificpromoter, and/or developmental specific promoter.

The nucleic acid that codes for a dominant negative protein can then beinserted into a cell or non-human animal. Any known method can be used.For example, stable transfection can be used. Additionally, a nucleicacid that codes for a dominant negative protein can be inserted into agenome of a non-human animal.

One or more genes in a non-human animal can be knocked out using anymethod known in the art. For example, knocking out one or more genes cancomprise deleting one or more genes from a genome of a non-human animal.Knocking out can also comprise removing all or a part of a gene sequencefrom a non-human animal. It is also contemplated that knocking out cancomprise replacing all or a part of a gene in a genome of a non-humananimal with one or more nucleotides. Knocking out one or more genes canalso comprise inserting a sequence in one or more genes therebydisrupting expression of the one or more genes. For example, inserting asequence can generate a stop codon in the middle of one or more genes.Inserting a sequence can also shift the open reading frame of one ormore genes.

Knockout can be done in any cell, organ, and/or tissue in a non-humananimal. For example, knockout can be whole body knockout, e.g.,expression of one or more genes is reduced in all cells of a non-humananimal. Knockout can also be specific to one or more cells, tissues,and/or organs of a non-human animal. This can be achieved by conditionalknockout, where expression of one or more genes is selectively reducedin one or more organs, tissues or types of cells. Conditional knockoutcan be performed by a Cre-lox system, where cre is expressed under thecontrol of a cell, tissue, and/or organ specific promoter. For example,one or more genes can be knocked out (or expression can be reduced) inone or more tissues, or organs, where the one or more tissues or organscan include brain, lung, liver, heart, spleen, pancreas, smallintestine, large intestine, skeletal muscle, smooth muscle, skin, bones,adipose tissues, hairs, thyroid, trachea, gall bladder, kidney, ureter,bladder, aorta, vein, esophagus, diaphragm, stomach, rectum, adrenalglands, bronchi, ears, eyes, retina, genitals, hypothalamus, larynx,nose, tongue, spinal cord, or ureters, uterus, ovary, testis, and/or anycombination thereof. One or more genes can also be knocked out (orexpression can be reduced) in one types of cells, where one or moretypes of cells include trichocytes, keratinocytes, gonadotropes,corticotropes, thyrotropes, somatotropes, lactotrophs, chromaffin cells,parafollicular cells, glomus cells melanocytes, nevus cells, merkelcells, odontoblasts, cementoblasts corneal keratocytes, retina mullercells, retinal pigment epithelium cells, neurons, glias (e.g.,oligodendrocyte astrocytes), ependymocytes, pinealocytes, pneumocytes(e.g., type I pneumocytes, and type II pneumocytes), clara cells, gobletcells, G cells, D cells, Enterochromaffin-like cells, gastric chiefcells, parietal cells, foveolar cells, K cells, D cells, I cells, gobletcells, paneth cells, enterocytes, microfold cells, hepatocytes, hepaticstellate cells (e.g., Kupffer cells from mesoderm), cholecystocytes,centroacinar cells, pancreatic stellate cells, pancreatic α cells,pancreatic β cells, pancreatic δ cells, pancreatic F cells, pancreatic εcells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroidchief cells), oxyphil cells, urothelial cells, osteoblasts, osteocytes,chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts,myocytes, myosatellite cells, tendon cells, cardiac muscle cells,lipoblasts, adipocytes, interstitial cells of cajal, angioblasts,endothelial cells, mesangial cells (e.g., intraglomerular mesangialcells and extraglomerular mesangial cells), juxtaglomerular cells,macula densa cells, stromal cells, interstitial cells, telocytes simpleepithelial cells, podocytes, kidney proximal tubule brush border cells,sertoli cells, leydig cells, granulosa cells, peg cells, germ cells,spermatozoon ovums, lymphocytes, myeloid cells, endothelial progenitorcells, endothelial stem cells, angioblasts, mesoangioblasts, pericytemural cells, and/or any combination thereof.

Conditional knockouts can be inducible, for example, by usingtetracycline inducible promoters, development specific promoters. Thiscan allow for eliminating or suppressing expression of a gene/protein atany time or at a specific time. For example, with the case of atetracycline inducible promoter, tetracycline can be given to anon-human animal any time after birth. If a non-human animal is a beingthat develops in a womb, then promoter can be induced by givingtetracycline to the mother during pregnancy. If a non-human animaldevelops in an egg, a promoter can be induced by injecting, orincubating in tetracycline. Once tetracycline is given to a non-humananimal, the tetracycline will result in expression of cre, which willthen result in excision of a gene of interest.

A cre/lox system can also be under the control of a developmentalspecific promoter. For example, some promoters are turned on afterbirth, or even after the onset of puberty. These promoters can be usedto control cre expression, and therefore can be used in developmentalspecific knockouts.

It is also contemplated that any combinations of knockout technology canbe combined. For example, tissue specific knockout can be combined withinducible technology, creating a tissue specific, inducible knockout.Furthermore, other systems such developmental specific promoter, can beused in combination with tissues specific promoters, and/or inducibleknockouts.

Knocking out technology can also comprise gene editing. For example,gene editing can be performed using a nuclease, including CRISPRassociated proteins (Cas proteins, e.g., Cas9), Zinc finger nuclease(ZFN), Transcription Activator-Like Effector Nuclease (TALEN), andmaganucleases. Nucleases can be naturally existing nucleases,genetically modified, and/or recombinant. For example, a CRISPR/cassystem can be suitable as a gene editing system.

It is also contemplated that less than all alleles of one or more genesof a non-human animal can be knocked out. For example, in diploidnon-human animals, it is contemplated that one of two alleles areknocked out. This can result in decreased expression and decreasedprotein levels of genes. Overall decreased expression can be less thanor less than about 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, or 20%; e.g., from or from about 99% to90%; 90% to 80%; 80% to 70%; 70% to 60%; 60% to 50%; 50% to 40%; 40% to30%, or 30% to 20%; compared to when both alleles are functioning, forexample, not knocked out and/or knocked down. Additionally, overalldecrease in protein level can be the same as the decreased in overallexpression. Overall decrease in protein level can be about or less thanabout 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%, e.g., from orfrom about 99% to 90%; 90% to 80%; 80% to 70%; 70% to 60%; 60% to 50%;50% to 40%; 40% to 30%, or 30% to 20%; compared to when both alleles arefunctioning, for example, not knocked out and/or knocked down. However,it is also contemplated that all alleles of one or more genes in anon-human animal can be knocked out.

Knocking out of one or more genes can be validated by genotyping.Methods for genotyping can include sequencing, restriction fragmentlength polymorphism identification (RFLPI), random amplified polymorphicdetection (RAPD), amplified fragment length polymorphism detection(AFLPD), PCR (e.g., long range PCR, or stepwise PCR), allele specificoligonucleotide (ASO) probes, and hybridization to DNA microarrays orbeads. For example, genotyping can be performed by sequencing. In somecases, sequencing can be high fidelity sequencing. Methods of sequencingcan include Maxam-Gilbert sequencing, chain-termination methods (e.g.,Sanger sequencing), shotgun sequencing, and bridge PCR. In some cases,genotyping can be performed by next-generation sequencing. Methods ofnext-generation sequencing can include massively parallel signaturesequencing, polony sequencing, pyrosequencing (e.g., pyrosequencingdeveloped by 454 Life Sciences), single-molecule rea-time sequencing(e.g., by Pacific Biosciences), Ion semiconductor sequencing (e.g., byIon Torrent semiconductor sequencing), sequencing by synthesis (e.g., bySolexa sequencing by Illumina), sequencing by ligation (e.g., SOLiDsequencing by Applied Biosystems), DNA nanoball sequencing, andheliscope single molecule sequencing. In some cases, genotyping of anon-human animal herein can comprise full genome sequencing analysis. Insome cases, knocking out of a gene in an animal can be validated bysequencing (e.g., next-generation sequencing) a part of the gene or theentire gene. For example, knocking out of NLRC5 gene in a pig can bevalidated by next generation sequencing of the entire NLRC5. The nextgeneration sequencing of NLRC5 can be performed using e.g. using forwardprimer 5′-gctgtggcatatggcagttc-3′ (SEQ ID No. 1) and reverse primer5′-tccatgtataagtctttta-3′ (SEQ ID No. 2), or forward primer5′-ggcaatgccagatcctcaac-3′ (SEQ ID No. 3) and reverse primer5′-tgtctgatgtctttctcatg-3′ (SEQ ID No. 4).

TABLE 1 Genomic sequences, cDNA and proteins of exemplary disruptedgenes Genomic sequence cDNA protein SEQ ID SEQ ID SEQ ID Gene No. No.Accession No. No. Accession No. NLRC5 5 6 KC514136.1 7 AGG68119.1 TAP1 89 NM_001044581 10 NP_001038046.1 GGTA1 11 12 AF221508 13 NP_998975.1CMAH 14 15 NM_001113015 16 NP_001106486.1 CXCL10 17 18 NM_001008691.1 19NP_001008691.1 CIITA 20 21 XM_013995652.1 22 XP_013851106.1 B4GALNT2 2324 NM_001244330.1 25 NP_001231259.1 C3 26 27 NM_214009.1 28 NP_999174.1MICA 29 30 NM_000247.2 31 NP_000238.1 MICB 32 33 NM_001289160.1 34NP_001276089.1Transgenes

Transgenes can be useful for overexpressing endogenous genes at higherlevels than without the transgenes. Additionally, transgenes can be usedto express exogenous genes. Transgenes can also encompass other types ofgenes, for example, a dominant negative gene.

A transgene of protein X can refer to a transgene comprising anucleotide sequence encoding protein X. As used herein, in some cases, atransgene encoding protein X can be a transgene encoding 100% or about100% of the amino acid sequence of protein X. In some cases, a transgeneencoding protein X can encode the full or partial amino sequence ofprotein X. For example, the transgene can encode at least or at leastabout 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%,e.g., from or from about 99% to 90%; 90% to 80%; 80% to 70%; 70% to 60%;or 60% to 50%; of the amino acid sequence of protein X. Expression of atransgene can ultimately result in a functional protein, e.g., apartially or fully functional protein. As discussed above, if a partialsequence is expressed, the ultimate result can be in some cases anonfunctional protein or a dominant negative protein. A nonfunctionalprotein or dominant negative protein can also compete with a functional(endogenous or exogenous) protein. A transgene can also encode an RNA(e.g., mRNA, shRNA, siRNA, or microRNA). In some cases, where atransgene encodes for an mRNA, this can in turn be translated into apolypeptide (e.g., a protein). Therefore, it is contemplated that atransgene can encode for protein. A transgene can, in some instances,encode a protein or a portion of a protein. Additionally, a protein canhave one or more mutations (e.g., deletion, insertion, amino acidreplacement, or rearrangement) compared to a wild-type polypeptide. Aprotein can be a natural polypeptide or an artificial polypeptide (e.g.,a recombinant polypeptide). A transgene can encode a fusion proteinformed by two or more polypeptides.

Transgenes can be placed into an organism, cell, tissue, or organ, in amanner which produces a product of the transgene. For example, disclosedherein is a non-human animal comprising one or more transgenes. One ormore transgenes can be in combination with one or more disruptions asdescribed herein. A transgene can be incorporated into a cell. Forexample, a transgene can be incorporated into an organism's germ line.When inserted into a cell, a transgene can be either a complementary DNA(cDNA) segment, which is a copy of messenger RNA (mRNA), or a geneitself residing in its original region of genomic DNA (with or withoutintrons).

A non-human animal can comprise one or more transgenes comprising one ormore polynucleotide inserts. The polynucleotide inserts can encode oneor proteins or functional fragments thereof. In some cases, a non-humananimal can comprise one or more transgenes comprising one or morepolynucleotide inserts encoding proteins that can reduce expressionand/or function of MHC molecules (e.g., MHC I molecules and/or MHC IImolecules). The one or more transgenes can comprise one or morepolynucleotide inserts encoding MHC I formation suppressors, regulatorsof complement activations, inhibitory ligands for NK cells, B7 familymembers, CD47, serine protease inhibitors, galectins, and/or anyfragments thereof. In some cases, the MHC I formation suppressors can beinfected cell protein 47 (ICP47). In some cases, regulators ofcomplement activation can comprise cluster of differentiation 46 (CD46),cluster of differentiation 55 (CD55), and cluster of differentiation 59(CD59). In some cases, inhibitory ligands for NK cells can compriseleukocyte antigen E (HLA-E), human leukocyte antigen G (HLA-G), andβ-2-microglobulin (B2M). An inhibitory ligand for NK cells can be anisoform of HLA-G, e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6,or HLA-G7. For example, inhibitory ligand for NK cells can be HLA-G1. Atransgene of HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5,HLA-G6, or HLA-G7) can refer to a transgene comprising a nucleotidesequence encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5,HLA-G6, or HLA-G7). As used herein, in some cases, a transgene encodingHLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7)can be a transgene encoding 100% or about 100% of the amino acidsequence of HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6,or HLA-G7). In other cases, a transgene encoding HLA-G (e.g., HLA-G1,HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7) can be a transgeneencoding the full or partial sequence of HLA-G (e.g., HLA-G1, HLA-G2,HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7). For example, the transgenecan encode at least or at least about 99%, 95%, 90%, 80%, 70%, 60%, or50% of the amino acid sequence of HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3,HLA-G4, HLA-G5, HLA-G6, or HLA-G7). For example, the transgene canencode 90% of the HLA-G amino acid sequence. A transgene can comprisepolynucleotides encoding a functional (e.g., a partially or fullyfunctional) HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6,or HLA-G7). In some cases, the one or more transgenes can comprise oneor more polynucleotide inserts encoding one or more of ICP47, CD46,CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5,HLA-G6, or HLA-G7), and B2M. The HLA-G genomic DNA sequence can have 8exons by which alternative splicing results in 7 isoforms. The HLA-G1isoform can exclude exon 7. The HLA-G2 isoform can exclude exon 3 and 7.Translation of intron 2 or intron 4 can result secreted isoforms due tothe loss of the transmembrane domain expression. The maps of the genomicsequence and cDNA of HLA-G are shown in FIGS. 14A-14B. In some cases, B7family members can comprise CD80, CD86, programed death-ligand 1(PD-L1), programed death-ligand 2 (PD-L2), CD275, CD276, V-set domaincontaining T cell activation inhibitor 1 (VTCN1), platelet receptorGi24, natural cytotoxicity triggering receptor 3 ligand 1 (NR3L1), andHERV-H LTR-associating 2 (HHLA2). For example, a B7 family member can bePD-L1 or PD-L2. In some cases, a serine protease inhibitor can be serineprotease inhibitor 9 (Spi9). In some cases, galectins can comprisegalectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6,galectin-7, galectin-8, galectin-9, galectin-10, galectin-11,galectin-12, galectin-13, galectin-14, and galectin-15. For example, agalectin can be galectin-9.

A genetically modified non-human animal can comprise reduced expressionof one or more genes and one or more transgenes disclosed herein. Insome cases, a genetically modified non-human animal can comprise reducedexpression of one or more of NLRC5, TAP1, CXCL10, MICA, MICB, C3, CIITA,GGTA1, CMAH, and B4GALNT2, and one or more transgenes comprising one ormore polynucleotide inserts encoding one or more of ICP47, CD46, CD55,CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5,HLA-G6, or HLA-G7), B2M, PD-L1, PD-L2, CD47, Spi9, and galectin-9. Insome cases, a genetically modified non-human animal can comprise reducedexpression GGTA1, CMAH, and B4GALNT2, and exogenous polynucleotidesencoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, orHLA-G7), CD47 (e.g., human CD47), PD-L1 (e.g., human PD-L1), and PD-L2(e.g., human PD-L2). In some cases, a genetically modified non-humananimal can comprise reduced expression GGTA1, CMAH, and B4GALNT2, andexogenous polynucleotides encoding HLA-E, CD47 (e.g., human CD47), PD-L1(e.g., human PD-L1), and PD-L2 (e.g., human PD-L2). In some cases, agenetically modified non-human animal can comprise reduced expressionNLRC5, C3, CXC10, GGTA1, CMAH, and B4GALNT2, and exogenouspolynucleotides encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4,HLA-G5, HLA-G6, or HLA-G7), CD47 (e.g., human CD47), PD-L1 (e.g., humanPD-L1), and PD-L2 (e.g., human PD-L2). In some cases, a geneticallymodified non-human animal can comprise reduced expression TAP1, C3,CXC10GGTA1, CMAH, and B4GALNT2, and exogenous polynucleotides encodingHLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7),CD47 (e.g., human CD47), PD-L1 (e.g., human PD-L1), and PD-L2 (e.g.,human PD-L2). In some cases, a genetically modified non-human animal cancomprise reduced expression NLRC5, C3, CXC10, GGTA1, CMAH, and B4GALNT2,and exogenous polynucleotides encoding HLA-E, CD47 (e.g., human CD47),PD-L1 (e.g., human PD-L1), and PD-L2 (e.g., human PD-L2). In some cases,a genetically modified non-human animal can comprise reduced expressionTAP1, C3, CXC10, GGTA1, CMAH, and B4GALNT2, and exogenouspolynucleotides encoding HLA-E. In some cases, a genetically modifiednon-human animal can comprise reduced expression of GGTA1 and atransgene comprising one or more polynucleotide inserts encoding HLA-E.In some cases, a genetically modified non-human animal can comprisereduced expression of GGTA1 and a transgene comprising one or morepolynucleotide inserts encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3,HLA-G4, HLA-G5, HLA-G6, or HLA-G7). In some cases, a geneticallymodified non-human animal can comprise a transgene comprising one ormore polynucleotide inserts encoding HLA-G (e.g., HLA-G1, HLA-G2,HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7) inserted adjacent to a Rosa26promoter, e.g., a porcine Rosa26 promoter. In some cases, a geneticallymodified non-human animal can comprise reduced expression of NLRC5, C3,GGTA1, CMAH, and B4GALNT2, and transgenes comprising polynucleotidesencoding proteins or functional fragments thereof, where the proteinscomprise HLA-G1, Spi9, PD-L1, PD-L2, CD47, and galectin-9. In somecases, a genetically modified non-human animal can comprise reducedexpression of TAP1, C3, GGTA1, CMAH, and B4GALNT2, and transgenescomprising polynucleotides encoding proteins or functional fragmentsthereof, where the proteins comprise HLA-G1, Spi9, PD-L1, PD-L2, CD47,and galectin-9. In some cases, a genetically modified non-human animalcan comprise reduced expression of NLRC5, TAP1, C3, GGTA1, CMAH, andB4GALNT2, and transgenes comprising polynucleotides encoding proteins orfunctional fragments thereof, where the proteins comprise HLA-G1, Spi9,PD-L1, PD-L2, CD47, and galectin-9. In some cases, a geneticallymodified non-human animal can comprise reduced protein expression ofNLRC5, C3, GGTA1, and CXCL10, and transgenes comprising polynucleotidesencoding proteins or functional fragments thereof, where the proteincomprise HLA-G1 or HLA-E. In some cases, a genetically modifiednon-human animal can comprise reduced protein expression of TAP1, C3,GGTA1, and CXCL10, and transgenes comprising polynucleotides encodingproteins or functional fragments thereof, where the protein compriseHLA-G1 or HLA-E. In some cases, a genetically modified non-human animalcan comprise reduced protein expression of NLRC5, TAP1, C3, GGTA1, andCXCL10, and transgenes comprising polynucleotides encoding proteins orfunctional fragments thereof, where the protein comprise HLA-G1 orHLA-E. In some cases, CD47, PD-L1, and PD-L2 encoded by the transgenesherein can be human CD47, human PD-L1 and human PD-L2.

A genetically modified non-human animal can comprise a transgeneinserted in a locus in the genome of the animal. In some cases, atransgene can be inserted adjacent to the promoter of or inside atargeted gene. In some cases, insertion of the transgene can reduce theexpression of the targeted gene. The targeted gene can be a gene whoseexpression is reduced disclosed herein. For example, a transgene can beinserted adjacent to the promoter of or inside one or more of NLRC5,TAP1, CXCL10, MICA, MICB, C3, CIITA, GGTA1, CMAH, and B4GALNT2. In somecases, a transgene can be inserted adjacent to the promoter of or insideGGTA1.

For example, a non-human animal can comprise one or more transgenescomprising one or more polynucleotide inserts of Infected cell protein47 (ICP47), Cluster of differentiation 46 (CD46), Cluster ofdifferentiation 55 (CD55), Cluster of differentiation 59 (CD 59), HLA-E,HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7),B2M, Spi9, PD-L1, PD-L2, CD47, galectin-9, any functional fragmentsthereof, or any combination thereof. Polynucleotide encoding for ICP47,CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4,HLA-G5, HLA-G6, or HLA-G7), or B2M can encode one or more of ICP47,CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4,HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47, or galectin-9human proteins. A non-human animal can comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more transgenes. Forexample, a non-human animal can comprise one or more transgenecomprising ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2,HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2,CD47, galectin-9, any functional fragments thereof, or any combinationthereof. A non-human animal can also comprise a single transgeneencoding ICP47. A non-human animal can sometimes comprise a singletransgene encoding CD59. A non-human animal can sometimes comprise asingle transgene encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4,HLA-G5, HLA-G6, or HLA-G7). A non-human animal can sometimes comprise asingle transgene encoding HLA-E. A non-human animal can sometimescomprise a single transgene encoding B2M. A non-human animal can alsocomprise two or more transgenes, where the two or more transgenes areICP47, CD46, CD55, CD59, and/or any combination thereof. For example,two or more transgenes can comprise CD59 and CD46 or CD59 and CD55. Anon-human animal can also comprise three or more transgenes, where thethree or more transgenes can comprise ICP47, CD46, CD55, CD59, or anycombination thereof. For example, three or more transgenes can compriseCD59, CD46, and CD55. A non-human animal can also comprise four or moretransgenes, where the four or more transgenes can comprise ICP47, CD46,CD55, and CD59. A non-human animal can comprise four or more transgenescomprising ICP47, CD46, CD55, and CD59.

A combination of transgenes and gene disruptions can be used. Anon-human animal can comprise one or more reduced genes and one or moretransgenes. For example, one or more genes whose expression is reducedcan comprise any one of NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10,MICA, MICB, C3, CIITA, and/or any combination thereof, and one or moretransgene can comprise ICP47, CD46, CD55, CD 59, any functionalfragments thereof, and/or any combination thereof. For example, solelyto illustrate various combinations, one or more genes whose expressionis disrupted can comprise NLRC5 and one or more transgenes compriseICP47. One or more genes whose expression is disrupted can also compriseTAP1, and one or more transgenes comprise ICP47. One or more genes whoseexpression is disrupted can also comprise NLRC5 and TAP1, and one ormore transgenes comprise ICP47. One or more genes whose expression isdisrupted can also comprise NLRC5, TAP1, and GGTA1, and one or moretransgenes comprise ICP47. One or more genes whose expression isdisrupted can also comprise NLRC5, TAP1, B4GALNT2, and CMAH, and one ormore transgenes comprise ICP47. One or more genes whose expression isdisrupted can also comprise NLRC5, TAP1, GGTA1, B4GALNT2, and CMAH, andone or more transgenes comprise ICP47. One or more genes whoseexpression is disrupted can also comprise NLRC5 and one or moretransgenes comprise CD59. One or more genes whose expression isdisrupted can also comprise TAP1, and one or more transgenes compriseCD59. One or more genes whose expression is disrupted can also compriseNLRC5 and TAP1, and one or more transgenes comprise CD59. One or moregenes whose expression is disrupted can also comprise NLRC5, TAP1, andGGTA1, and one or more transgenes comprise CD59. One or more genes whoseexpression is disrupted can also comprise NLRC5, TAP1, B4GALNT2, andCMAH, and one or more transgenes comprise CD59. One or more genes whoseexpression is disrupted can also comprise NLRC5, TAP1, GGTA1, B4GALNT2,and CMAH, and one or more transgenes comprise CD59.

Transgenes that can be used and are specifically contemplated caninclude those genes that exhibit a certain identity and/or homology togenes disclosed herein, for example, ICP47, CD46, CD55, CD59, HLA-E,HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7),B2M, Spi9, PD-L1, PD-L2, CD47, galectin-9, any functional fragmentsthereof, and/or any combination thereof. Therefore, it is contemplatedthat if gene that exhibits at least or at least about 60%, 70%, 80%,90%, 95%, 98%, or 99% homology, e.g., at least or at least about 99% to90%; 90% to 80%; 80% to 70%; 70% to 60% homology; (at the nucleic acidor protein level), it can be used as a transgene. It is alsocontemplated that a gene that exhibits at least or at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, identity e.g., at leastor at least about 99% to 90%; 90% to 80%; 80% to 70%; 70% to 60%identity; (at the nucleic acid or protein level) can be used as atransgene.

A non-human animal can also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more dominant negativetransgenes. Expression of a dominant negative transgenes can suppressexpression and/or function of a wild type counterpart of the dominantnegative transgene. Thus, for example, a non-human animal comprising adominant negative transgene X, can have similar phenotypes compared to adifferent non-human animal comprising an X gene whose expression isreduced. One or more dominant negative transgenes can be dominantnegative NLRC5, dominant negative TAP1, dominant negative GGTA1,dominant negative CMAH, dominant negative B4GALNT2, dominant negativeCXCL10, dominant negative MICA, dominant negative MICB, dominantnegative CIITA, dominant negative C3, or any combination thereof.

Also provided is a non-human animal comprising one or more transgenesthat encodes one or more nucleic acids that can suppress geneticexpression, e.g., can knockdown a gene. RNAs that suppress geneticexpression can comprise, but are not limited to, shRNA, siRNA, RNAi, andmicroRNA. For example, siRNA, RNAi, and/or microRNA can be given to anon-human animal to suppress genetic expression. Further, a non-humananimal can comprise one or more transgene encoding shRNAs. shRNA can bespecific to a particular gene. For example, a shRNA can be specific toany gene described in the application, including but not limited to,NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, B4GALNT2, CIITA,C3, and/or any combination thereof.

When transplanted to a subject, cells, tissues, or organs from thegenetically modified non-human animal can trigger lower immune responses(e.g., transplant rejection) in the subject compared to cells, tissues,or organs from a non-genetically modified counterpart. In some cases,the immune responses can include the activation, proliferation andcytotoxicity of T cells (e.g., CD8+ T cells and/or CD4+ T cells) and NKcells. Thus, phenotypes of genetically modified cells disclosed hereincan be measured by co-culturing the cells with NK cells, T cells (e.g.,CD8+ T cells or CD4+ T cells), and testing the activation, proliferationand cytotoxicity of the NK cells or T cells. In some cases, the T cellsor NK cells activation, proliferation and cytotoxicity induced by thegenetically modified cells can be lower than that induced bynon-genetically modified cells. In some cases, phenotypes of geneticallymodified cells herein can be measured by Enzyme-Linked ImmunoSpot(ELISPOT) assays.

One or more transgenes can be from different species. For example, oneor more transgenes can comprise a human gene, a mouse gene, a rat gene,a pig gene, a bovine gene, a dog gene, a cat gene, a monkey gene, achimpanzee gene, or any combination thereof. For example, a transgenecan be from a human, having a human genetic sequence. One or moretransgenes can comprise human genes. In some cases, one or moretransgenes are not adenoviral genes.

A transgene can be inserted into a genome of a non-human animal in arandom or site-specific manner. For example, a transgene can be insertedto a random locus in a genome of a non-human animal. These transgenescan be fully functional if inserted anywhere in a genome. For instance,a transgene can encode its own promoter or can be inserted into aposition where it is under the control of an endogenous promoter.Alternatively, a transgene can be inserted into a gene, such as anintron of a gene or an exon of a gene, a promoter, or a non-codingregion.

Sometimes, more than one copy of a transgene can be inserted into morethan a random locus in a genome. For example, multiple copies can beinserted into a random locus in a genome. This can lead to increasedoverall expression than if a transgene was randomly inserted once.Alternatively, a copy of a transgene can be inserted into a gene, andanother copy of a transgene can be inserted into a different gene. Atransgene can be targeted so that it could be inserted to a specificlocus in a genome of a non-human animal.

Expression of a transgene can be controlled by one or more promoters. Apromoter can be a ubiquitous, tissue-specific promoter or an induciblepromoter. Expression of a transgene that is inserted adjacent to apromoter can be regulated. For example, if a transgene is inserted nearor next to a ubiquitous promoter, the transgene will be expressed in allcells of a non-human animal. Some ubiquitous promoters can be a CAGGSpromoter, an hCMV promoter, a PGK promoter, an SV40 promoter, or aRosa26 promoter.

A promoter can be endogenous or exogenous. For example, one or moretransgenes can be inserted adjacent to an endogenous or exogenous Rosa26promoter. Further, a promoter can be specific to a non-human animal. Forexample, one or more transgenes can be inserted adjacent to a porcineRosa26 promoter.

Tissue specific promoter (which can be synonymous with cell-specificpromoters) can be used to control the location of expression. Forexample, one or more transgenes can be inserted adjacent to atissue-specific promoter. Tissue-specific promoters can be a FABPpromoter, a Lck promoter, a CamKII promoter, a CD19 promoter, a Keratinpromoter, an Albumin promoter, an aP2 promoter, an insulin promoter, anMCK promoter, an MyHC promoter, a WAP promoter, or a Col2A promoter. Forexample, a promoter can be a pancreas-specific promoter, e.g., aninsulin promoter.

Inducible promoters can be used as well. These inducible promoters canbe turned on and off when desired, by adding or removing an inducingagent. It is contemplated that an inducible promoter can be a Lac, tac,trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA,T7, VHB, Mx, and/or Trex.

A non-human animal or cells as described herein can comprise a transgeneencoding insulin. A transgene encoding insulin can be a human gene, amouse gene, a rat gene, a pig gene, a cattle gene, a dog gene, a catgene, a monkey gene, a chimpanzee gene, or any other mammalian gene. Forexample, a transgene encoding insulin can be a human gene. A transgeneencoding insulin can also be a chimeric gene, for example, a partiallyhuman gene.

Expression of transgenes can be measured by detecting the level oftranscripts of the transgenes. For example, expression of transgenes canbe measured by Northern blotting, nuclease protection assays (e.g.,RNase protection assays), reverse transcription PCR, quantitative PCR(e.g., real-time PCR such as real-time quantitative reversetranscription PCR), in situ hybridization (e.g., fluorescent in situhybridization (FISH)), dot-blot analysis, differential display, Serialanalysis of gene expression, subtractive hybridization, microarrays,nanostring, and/or sequencing (e.g., next-generation sequencing). Insome cases, expression of transgenes can be measured by detectingproteins encoded by the genes. For example, expression of one or moregenes can be measured by protein immunostaining, proteinimmunoprecipitation, electrophoresis (e.g., SDS-PAGE), Western blotting,bicinchoninic acid assay, spectrophotometry, mass spectrometry, enzymeassays (e.g., enzyme-linked immunosorbent assays), immunohistochemistry,flow cytometry, and/or immunocytochemistry. In some cases, expression oftransgenes can be measured by microscopy. The microscopy can be optical,electron, or scanning probe microscopy. In some cases, opticalmicroscopy comprises use of bright field, oblique illumination,cross-polarized light, dispersion staining, dark field, phase contrast,differential interference contrast, interference reflection microscopy,fluorescence (e.g., when particles, e.g., cells, are immunostained),confocal, single plane illumination microscopy, light sheet fluorescencemicroscopy, deconvolution, or serial time-encoded amplified microscopy.

Insertion of transgenes can be validated by genotyping. Methods forgenotyping can include sequencing, restriction fragment lengthpolymorphism identification (RFLPI), random amplified polymorphicdetection (RAPD), amplified fragment length polymorphism detection(AFLPD), PCR (e.g., long range PCR, or stepwise PCR), allele specificoligonucleotide (ASO) probes, and hybridization to DNA microarrays orbeads. In some cases, genotyping can be performed by sequencing. In somecases, sequencing can be high fidelity sequencing. Methods of sequencingcan include Maxam-Gilbert sequencing, chain-termination methods (e.g.,Sanger sequencing), shotgun sequencing, and bridge PCR. In some cases,genotyping can be performed by next-generation sequencing. Methods ofnext-generation sequencing can include massively parallel signaturesequencing, polony sequencing, pyrosequencing (e.g., pyrosequencingdeveloped by 454 Life Sciences), single-molecule rea-time sequencing(e.g., by Pacific Biosciences), Ion semiconductor sequencing (e.g., byIon Torrent semiconductor sequencing), sequencing by synthesis (e.g., bySolexa sequencing by Illumina), sequencing by ligation (e.g., SOLiDsequencing by Applied Biosystems), DNA nanoball sequencing, andheliscope single molecule sequencing. In some cases, genotyping of anon-human animal herein can comprise full genome sequencing analysis.

In some cases, insertion of a transgene in an animal can be validated bysequencing (e.g., next-generation sequencing) a part of the transgene orthe entire transgene. For example, insertion of a transgene adjacent toa Rosa26 promoter in a pig can be validated by next generationsequencing of Rosa exons 1 to 4, e.g., using the forward primer5′-cgcctagagaagaggctgtg-3′ (SEQ ID No. 35), and reverse primer5′-ctgctgtggctgtggtgtag-3′ (SEQ ID No. 36).

TABLE 2 cDNA sequences of exemplary transgenes SEQ ID No. Gene AccessionNo. 37 CD46 NM_213888 38 CD55 AF228059.1 39 CD59 AF020302 40 ICP47EU445532.1 41 HLA-G1 NM_002127.5 42 HLA-E NM_005516.5 43 Human β-2-NM_004048.2 microglobulin 44 Human PD-L1 NM_001267706.1 45 Human PD-L2NM_025239.3 46 Human Spi9 NM_004155.5 47 Human CD47 NM_001777.3 48 Humangalectin-9 NM_009587.2

TABLE 3 Sequences of proteins encoded by exemplary transgenes SEQ ID No.Protein Accession No. 49 CD46 NP_999053.1 50 CD55 AAG14412.1 51 CD59AAC67231.1 52 ICP47 ACA28836.1 53 HLA-G1 NP_002118.1 54 HLA-ENP_005507.3 55 Human β-2- NP_004039.1 microglobulin 56 Human PD-L1NP_001254635.1 57 Human PD-L2 NP_079515.2 58 Human Spi9 NP_004146.1 59Human CD47 NP_001768.1 60 Human galectin-9 NP_033665.1Populations of Non-Human Animals

Provided herein is a single non-human animal and also a population ofnon-human animals. A population of non-human animals can be geneticallyidentical. A population of non-human animals can also be phenotypicalidentical. A population of non-human animals can be both phenotypicaland genetically identical.

Further provided herein is a population of non-human animals, which canbe genetically modified. For example, a population can comprise at leastor at least about 2, 5, 10, 50, 100, or 200, non-human animals asdisclosed herein. The non-human animals of a population can haveidentical phenotypes. For example, the non-human animals of a populationcan be clones. A population of non-human animal can have identicalphysical characteristics. The non-human animals of a population havingidentical phenotypes can comprise a same transgene(s). The non-humananimals of a population having identical phenotypes can also comprise asame gene(s) whose expression is reduced. The non-human animals of apopulation having identical phenotypes can also comprise a same gene(s)whose expression is reduced and comprise a same transgene(s). Apopulation of non-human animals can comprise at least or at least about2, 5, 10, 50, 100, or 200, non-human animals having identicalphenotypes. For example, the phenotypes of any particular litter canhave the identical phenotype (e.g., in one example, anywhere from 1 toabout 20 non-human animals). The non-human animals of a population canbe pigs having identical phenotypes.

The non-human animals of a population can have identical genotypes. Forexample, all nucleic acid sequences in the chromosomes of non-humananimals in a population can be identical. The non-human animals of apopulation having identical genotypes can comprise a same transgene(s).The non-human animals of a population having identical genotypes canalso comprise a same gene(s) whose expression is reduced. The non-humananimals of a population having identical genotypes can also comprise asame gene(s) whose expression is reduced and comprise a sametransgene(s). A population of non-human animals can comprise at least orat least about 2, 5, 50, 100, or 200 non-human animals having identicalgenotypes. The non-human animals of a population can be pigs havingidentical genotypes.

Cells from two or more non-human animals with identical genotypes and/orphenotypes can be used in a tolerizing vaccine. In some cases, atolerizing vaccine disclosed herein can comprise a plurality of thecells (e.g., genetically modified cells) from two or more non-humananimals (e.g., pigs) with identical genotypes and/or phenotypes. Amethod for immunotolerizing a recipient to a graft can compriseadministering to the recipient a tolerizing vaccine comprising aplurality of cells (e.g., genetically modified cells) from two or morenon-human animals with identical genotypes or phenotypes.

Cells from two or more non-human animals with identical genotypes and/orphenotypes can be used in transplantation. In some cases, a graft (e.g.,xenograft or allograft) can comprise a plurality of cells from two ormore non-human animals with identical genotypes and/or phenotypes. Inembodiments of the methods described herein, e.g., a method for treatinga disease in a subject in need thereof, can comprise transplanting aplurality of cells (e.g., genetically modified cells) from two or morenon-human animals with identical genotypes and/or phenotypes.

Populations of non-human animals can be generated using any method knownin the art. In some cases, populations of non-human animals can begenerated by breeding. For example, inbreeding can be used to generate aphenotypically or genetically identical non-human animal or populationof non-human animals. Inbreeding, for example, sibling to sibling orparent to child, or grandchild to grandparent, or great grandchild togreat grandparent, can be used. Successive rounds of inbreeding caneventually produce a phenotypically or genetically identical non-humananimal. For example, at least or at least about 2, 3, 4, 5, 10, 20, 30,40, or 50 generations of inbreeding can produce a phenotypically and/ora genetically identical non-human animal. It is thought that after 10-20generations of inbreeding, the genetic make-up of a non-human animal isat least 99% pure. Continuous inbreeding can lead to a non-human animalthat is essentially isogenic, or close to isogenic as a non-human animalcan be without being an identical twin.

Breeding can be performed using non-human animals that have the samegenotype. For example, the non-human animals have the same gene(s) whoseexpression is reduced and/or carry the same transgene(s). Breeding canalso be performed using non-human animals having different genotypes.Breeding can be performed using a genetically modified non-human animaland non-genetically modified non-human animal, for example, agenetically modified female pig and a wild-type male pig, or agenetically modified male pig and a wild-type female pig. All thesecombinations of breeding can be used to produce a non-human animal ofdesire.

Populations of genetically modified non-human animals can also begenerated by cloning. For example, the populations of geneticallymodified non-human animal cells can be asexually producing similarpopulations of genetically or phenotypically identical individualnon-human animals. Cloning can be performed by various methods, such astwinning (e.g., splitting off one or more cells from an embryo and growthem into new embryos), somatic cell nuclear transfer, or artificialinsemination. More details of the methods are provided throughout thedisclosure.

II. Genetically Modified Cells

Disclosed herein are one or more genetically modified cells that can beused to treat or prevent disease. These genetically modified cells canbe from genetically modified non-human animals. For example, geneticallymodified non-human animals as disclosed above can be processed so thatone or more cells are isolated to produce isolated genetically modifiedcells. These isolated cells can also in some cases be furthergenetically modified cells. However, a cell can be modified ex vivo,e.g., outside an animal using modified or non-modified human ornon-human animal cells. For example, cells (including human andnon-human animal cells) can be modified in culture. It is alsocontemplated that a genetically modified cell can be used to generate agenetically modified non-human animal described herein. In some cases,the genetically modified cell can be isolated from a geneticallymodified animal. In some cases, the genetically modified cell can bederived from a cell from a non-genetically modified animal. Isolation ofcells can be performed by methods known in the art, including methods ofprimary cell isolation and culturing. It is specifically contemplatedthat a genetically modified cell is not extracted from a human.

Therefore, anything that can apply to the genetically modified non-humananimals including the various methods of making as described throughoutcan also apply herein. For example, all the genes that are disrupted andthe transgenes that are overexpressed are applicable in makinggenetically modified cells used herein. Further, any methods for testingthe genotype and expression of genes in the genetically modifiednon-human animals described throughout can be used to test the geneticmodification of the cells.

A genetically modified cell can be from a member of the Laurasiatheriasuperorder or a non-human primate. Such genetically modified cell can beisolated from a member of the Laurasiatheria superorder or a non-humanprimate. Alternatively, such genetically modified cell can be originatedfrom a member of the Laurasiatheria superorder or a non-human primate.For example, the genetically modified cell can be made from a cellisolated from a member of the Laurasiatheria superorder or a non-humanprimate, e.g., using cell culturing or genetic modification methods.

Genetically modified cells, e.g., cells from a genetically modifiedanimal or cells made ex vivo, can be analyzed and sorted. In some cases,genetically modified cells can be analyzed and sorted by flow cytometry,e.g., fluorescence-activated cell sorting. For example, geneticallymodified cells expressing a transgene can be detected and purified fromother cells using flow cytometry based on a label (e.g., a fluorescentlabel) recognizing the polypeptide encoded by the transgene.

Stem cells, including, non-human animal and human stem cells can beused. Stem cells do not have the capability to generating a viable humanbeing. For example, stem cells can be irreversibly differentiated sothat they are unable to generate a viable human being. Stem cells can bepluripotent, with the caveat that the stem cells cannot generate aviable human.

As discussed above in the section regarding the genetically modifiednon-human animals, the genetically modified cells can comprise one ormore genes whose expression is reduced. The same genes as disclosedabove for the genetically modified non-human animals can be disrupted.For example, a genetically modified cell comprising one or more geneswhose expression is disrupted, e.g., reduced, where the one or moregenes comprise NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB,C3, CIITA and/or any combination thereof. Further, the geneticallymodified cell can comprise one or more transgenes comprising one or morepolynucleotide inserts. For example, a genetically modified cell cancomprise one or more transgenes comprising one or more polynucleotideinserts of ICP47, CD46, CD55, CD 59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2,HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2,CD47, galectin-9, any functional fragments thereof, or any combinationthereof. A genetically modified cell can comprise one or more reducedgenes and one or more transgenes. For example, one or more genes whoseexpression is reduced can comprise any one of NLRC5, TAP1, GGTA1,B4GALNT2, CMAH, CXCL10, MICA, MICB, CIITA, and/or any combinationthereof, and one or more transgene can comprise ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6,or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47, galectin-9, any functionalfragments thereof, and/or any combination thereof. In some cases, agenetically modified cell can comprise reduced expression of NLRC5, C3,GGTA1, CMAH, and B4GALNT2, and transgenes comprising polynucleotidesencoding proteins or functional fragments thereof, where the proteinscomprise HLA-G1, Spi9, PD-L1, PD-L2, CD47, and galectin-9. In somecases, a genetically modified cell can comprise reduced expression ofTAP1, C3, GGTA1, CMAH, and B4GALNT2, and transgenes comprisingpolynucleotides encoding proteins or functional fragments thereof, wherethe proteins comprise HLA-G1, Spi9, PD-L1, PD-L2, CD47, and galectin-9.In some cases, a genetically modified cell can comprise reducedexpression of NLRC5, TAP1, C3, GGTA1, CMAH, and B4GALNT2, and transgenescomprising polynucleotides encoding proteins or functional fragmentsthereof, where the proteins comprise HLA-G1, Spi9, PD-L1, PD-L2, CD47,and galectin-9. In some cases, CD47, PD-L1, and PD-L2 encoded by thetransgenes herein can be human CD47, human PD-L1 and human PD0-L2. Insome cases, the genetically modified cell can be coated with CD47 on itssurface. Coating of CD47 on the surface of a cell can be accomplished bybiotinylating the cell surface followed by incubating the biotinylatedcell with a streptavidin-CD47 chimeric protein. The coated CD47 can behuman CD47.

As discussed above in the section regarding the genetically modifiednon-human animals, the genetically modified cell can comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moredisrupted genes. A genetically modified cell can also comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or moretransgenes.

As discussed in detail above, a genetically modified cell, e.g., porcinecell, can also comprise dominant negative transgenes and/or transgenesexpressing one or more knockdown genes. Also as discussed above,expression of a transgene can be controlled by one or more promoters.

A genetically modified cell can be one or more cells from tissues ororgans, the tissues or organs including brain, lung, liver, heart,spleen, pancreas, small intestine, large intestine, skeletal muscle,smooth muscle, skin, bones, adipose tissues, hairs, thyroid, trachea,gall bladder, kidney, ureter, bladder, aorta, vein, esophagus,diaphragm, stomach, rectum, adrenal glands, bronchi, ears, eyes, retina,genitals, hypothalamus, larynx, nose, tongue, spinal cord, or ureters,uterus, ovary and testis. For example, a genetically modified cell,e.g., porcine cell, can be from brain, heart, liver, skin, intestine,lung, kidney, eye, small bowel, or pancreas. In some cases, agenetically modified cell can be from a pancreas. More specifically,pancreas cells can be islet cells. Further, one or more cells can bepancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic Fcells (e.g., PP cells), or pancreatic ε cells. For example, agenetically modified cell can be pancreatic β cells. Tissues or organsdisclosed herein can comprise one or more genetically modified cells.The tissues or organs can be from one or more genetically modifiedanimals described in the application, e.g., pancreatic tissues such aspancreatic islets from one or more genetically modified pigs.

A genetically modified cell, e.g., porcine cell, can comprise one ormore types of cells, where the one or more types of cells includeTrichocytes, keratinocytes, gonadotropes, corticotropes, thyrotropes,somatotropes, lactotrophs, chromaffin cells, parafollicular cells,glomus cells melanocytes, nevus cells, merkel cells, odontoblasts,cementoblasts corneal keratocytes, retina muller cells, retinal pigmentepithelium cells, neurons, glias (e.g., oligodendrocyte astrocytes),ependymocytes, pinealocytes, pneumocytes (e.g., type I pneumocytes, andtype II pneumocytes), clara cells, goblet cells, G cells, D cells, ECLcells, gastric chief cells, parietal cells, foveolar cells, K cells, Dcells, I cells, goblet cells, paneth cells, enterocytes, microfoldcells, hepatocytes, hepatic stellate cells (e.g., Kupffer cells frommesoderm), cholecystocytes, centroacinar cells, pancreatic stellatecells, pancreatic α cells, pancreatic β cells, pancreatic δ cells,pancreatic F cells (e.g., PP cells), pancreatic ε cells, thyroid (e.g.,follicular cells), parathyroid (e.g., parathyroid chief cells), oxyphilcells, urothelial cells, osteoblasts, osteocytes, chondroblasts,chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tendon cells, cardiac muscle cells, lipoblasts,adipocytes, interstitial cells of cajal, angioblasts, endothelial cells,mesangial cells (e.g., intraglomerular mesangial cells andextraglomerular mesangial cells), juxtaglomerular cells, macula densacells, stromal cells, interstitial cells, telocytes simple epithelialcells, podocytes, kidney proximal tubule brush border cells, sertolicells, leydig cells, granulosa cells, peg cells, germ cells,spermatozoon ovums, lymphocytes, myeloid cells, endothelial progenitorcells, endothelial stem cells, angioblasts, mesoangioblasts, andpericyte mural cells. A genetically modified cell can potentially be anycells used in cell therapy. For example, cell therapy can be pancreaticβ cells supplement or replacement to a disease such as diabetes.

A genetically modified cell, e.g., porcine cell, can be from (e.g.,extracted from) a non-human animal. One or more cells can be from amature adult non-human animal. However, one or more cells can be from afetal or neonatal tissue.

Depending on the disease, one or more cells can be from a transgenicnon-human animal that has grown to a sufficient size to be useful as anadult donor, e.g., an islet cell donor. In some cases, non-human animalscan be past weaning age. For example, non-human animals can be at leastor at least about six months old. In some cases, non-human animals canbe at least or at least about 18 months old. A non-human animal in somecases, survive to reach breeding age. For example, islets forxenotransplantation can be from neonatal (e.g., age 3-7 days) orpre-weaning (e.g., age 14 to 21 days) donor pigs. One or moregenetically modified cells, e.g., porcine cells, can be cultured cells.For example, cultured cells can be from wild-type cells or fromgenetically modified cells (as described herein). Furthermore, culturedcells can be primary cells. Primary cells can be extracted and frozen,e.g., in liquid nitrogen or at −20° C. to −80° C. Cultured cells canalso be immortalized by known methods, and can be frozen and stored,e.g., in liquid nitrogen or at −20° C. to −80° C.

Genetically modified cells, e.g., porcine cells, as described herein canhave a lower risk of rejection, when compared to when a wild-typenon-genetically modified cell is transplanted.

Disclosed herein is a vector comprising a polynucleotide sequence ofICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3,HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47,galectin-9, any functional fragments thereof, or any combinationthereof. These vectors can be inserted into a genome of a cell (bytransfection, transformation, viral delivery, or any other knownmethod). These vectors can encode ICP47, CD46, CD55, CD59, HLA-E, HLA-G(e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2MSpi9, PD-L1, PD-L2, CD47, and/or galectin-9 proteins or functionalfragments thereof.

Vectors contemplated include, but not limited to, plasmid vectors,artificial/mini-chromosomes, transposons, and viral vectors. Furtherdisclosed herein is an isolated or synthetic nucleic acid comprising anRNA, where the RNA is encoded by any sequence in Table 2. RNA can alsoencode for any sequence that exhibits at least or at least about 50%,60%, 70%, 80%, 90%, 95%, 99%, or 100% homology to any sequence in Table2. RNA can also encode for any sequence that exhibits at least or atleast about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identity to anysequence in Table 2.

RNA can be a single-chain guide RNA. The disclosure also provides anisolated or synthesized nucleic acid comprising any sequence in Table 1.RNA can also provide an isolated or synthesized nucleic acid thatexhibits at least or at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%,or 100% homology to any sequence in Table 1. RNA can also provide anisolated or synthesized nucleic acid that exhibits at least or at leastabout 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identity to anysequence in Table 1.

Guide RNA sequences can be used in targeting one or more genes in agenome of a non-human animal. For example, guide RNA sequence can targeta single gene in a genome of non-human animal. In some cases, guide RNAsequences can target one or more target sites of each of one or moregenes in a genome of a non-human animal.

Genetically modified cells can also be leukocytes, lymphocytes, Blymphocytes, or any other cell such as islet cells, islet beta cells, orhepatocytes. These cells can be fixed or made apopototic by any methoddisclosed herein, e.g., by ECDI fixation.

A genetically modified cells can be derived (e.g., retrieved) from anon-human fetal animal, perinatal non-human animal, neonatal non-humananimal, preweaning non-human animal, young adult non-human animal, adultnon-human animal, or any combination thereof. In some cases, agenetically modified non-human animal cell can be derived from anembryonic tissue, e.g., an embryonic pancreatic tissue. For example, agenetically modified cell can be derived (e.g., retrieved) from anembryonic pig pancreatic tissue from embryonic day 42 (E42).

The term “fetal animal” and its grammatical equivalents can refer to anyunborn offspring of an animal. The term “perinatal animal” and itsgrammatical equivalents can refer to an animal immediately before orafter birth. For example, a perinatal period can start from 20th to 28thweek of gestation and ends 1 to 4 weeks after birth. The term “neonatalanimal” and its grammatical equivalents can refer to any new bornanimals. For example, a neonatal animal can be an animal born within amonth. The term “preweaning non-human animal” and its grammaticalequivalents can refer to any animal before being withdrawn from themother's milk.

Genetically modified non-human animal cells can be formulated into apharmaceutical composition. For example, the genetically modifiednon-human animal cells can be combined with a pharmaceuticallyacceptable excipient. An excipient that can be used is saline. Thepharmaceutical composition can be used to treat patients in need oftransplantation.

A genetically modified cell can comprise reduced expression of anygenes, and/or any transgenes disclosed herein. Genetic modification ofthe cells can be done by using any of the same method as describedherein for making the genetically modified animals. In some cases, amethod of making a genetically modified cell originated from a non-humananimal can comprise reducing expression of one or more genes and/orinserting one or more transgenes. The reduction of gene expressionand/or transgene insertion can be performed using any methods describedin the application, e.g., gene editing.

Genetically Modified Cells Derived from Stem Cells

Genetically modified cells can be a stem cell. These geneticallymodified stem cells can be used to make a potentially unlimited supplyof cells that can be subsequently processed into fixed or apoptoticcells by the methods disclosed herein. As discussed above, stem cellsare not capable of generating a viable human being.

The production of hundreds of millions of insulin-producing,glucose-responsive pancreatic beta cells from human pluripotent stemcells provides an unprecedented cell source for cell transplantationtherapy in diabetes (Pagliuca et al., 2014). Other human stemcell-(embryonic, pluripotent, placental, induced pluripotent, etc.)derived cell sources for cell transplantation therapy in diabetes and inother diseases are being developed.

These stem cell-derived cellular grafts are subject to rejection. Therejection can be mediated by CD8+ T cells. In Type 1 diabeticrecipients, human stem cell-derived functional beta cells are subject torejection and autoimmune recurrence. Both are thought to be mediated byCD8+ T cells.

To interfere with activation and effector function of theseallo-reactive and auto-reactive CD8+ T cells, established molecularmethods of gene modification, including CRISP/Cas9 gene targeting, canbe used to mutate the NLRC5, TAP1, and/or B2M genes in human stem cellsfor the purpose of preventing cell surface expression of functional MHCclass I in the stem cell-derived, partially or fully differentiatedcellular graft. Thus, transplanting human stem cell-derived cellulargrafts lacking functional expression of MHC class I can minimize therequirements of immunosuppression otherwise required to preventrejection and autoimmune recurrence.

However, lack of MHC class I expression on transplanted human cells willlikely cause the passive activation of natural killer (NK) cells (Ohlenet al, 1989). NK cell cytotoxicity can be overcome by the expression ofthe human MHC class 1 gene, HLA-E, which stimulates the inhibitoryreceptor CD94/NKG2A on NK cells to prevent cell killing (Weiss et al.,2009; Lilienfeld et al., 2007; Sasaki et al., 1999). Successfulexpression of the HLA-E gene was dependent on co-expression of the humanB2M (beta 2 microglobulin) gene and a cognate peptide (Weiss et al.,2009; Lilienfeld et al., 2007; Sasaki et al., 1999; Pascasova et al.,1999). A nuclease mediated break in the stem cell DNA allows for theinsertion of one or multiple genes via homology directed repair. TheHLA-E and hB2M genes in series can be integrated in the region of thenuclease mediated DNA break thus preventing expression of the targetgene (for example, NLRC5) while inserting the transgenes.

To further minimize, if not eliminate, the need for maintenanceimmunosuppression in recipients of stem cell derived cellular graftslacking functional expression of MHC class I, recipients of these graftscan also be treated with tolerizing apoptotic donor cells disclosedherein.

The methods for the production of insulin-producing pancreatic betacells (Pagliuca et al., 2014) can potentially be applied to non-human(e.g., pig) primary isolated pluripotent, embryonic stem cells orstem-like cells (Goncalves et al., 2014; Hall et al. V. 2008). However,the recipient of these insulin-producing pancreatic beta cells likelyhas an active immune response that threatens the success of the graft.To overcome antibody-mediated and CD8+ T cell immune attack, the donoranimal can be genetically modified before isolation of primary non-humanpluripotent, embryonic stem cells or stem-like cells to prevent theexpression of the GGTA1, CMAH, B4GalNT2, or MHC class I-related genes asdisclosed throughout the application. The pluripotent, embryonic stemcells or stem-like cells isolated from genetically modified animalscould then be differentiated into millions of insulin-producingpancreatic beta cells.

Xenogeneic stem cell-derived cell transplants can be desirable in somecases. For example, the use of human embryonic stem cells may beethically objectionable to the recipient. Therefore, human recipientsmay feel more comfortable receiving a cellular graft derived fromnon-human sources of embryonic stem cells.

Non-human stem cells may include pig stem cells. These stem cells can bederived from wild-type pigs or from genetically engineered pigs. Ifderived from wild-type pigs, genetic engineering using establishedmolecular methods of gene modification, including CRISP/Cas9 genetargeting, may best be performed at the stem cell stage. Geneticengineering may be targeted to disrupt expression of NLRC5, TAP1, and/orB2M genes to prevent functional expression of MHC class I. Disruptinggenes such as NLRC5, TAP1, and B2M in the grafts can cause lack offunctional expression of MHC class I on graft cells including on isletbeta cells, thereby interfering with the post-transplant activation ofautoreactive CD8+ T cells. Thus, this can protect the transplant, e.g.,transplanted islet beta cells, from the cytolytic effector functions ofautoreactive CD8+ T cells.

However, as genetic engineering of stem cells may alter their potentialfor differentiation, an approach can be to generate stem cell lines fromgenetically engineered pigs, including those pigs, in whom theexpression of NLRC5, TAP1, and/or B2M genes has been disrupted.

Generation of stem cells from pigs genetically modified to prevent theexpression also of the GGTA1, CMAH, B4GalNT2 genes or modified toexpress transgenes that encode for complement regulatory proteins CD46,CD55, or CD59, as disclosed throughout the application, could furtherimprove the therapeutic use of the insulin-producing pancreatic betacells or other cellular therapy products. Likewise, the same strategy asdescribed herein can be used in other methods and compositions describedthroughout.

Like in recipients of human stem cell-derived cellular grafts lackingfunctional expression of MHC class I, the need for maintenanceimmunosuppression in recipients of pig stem cell-derived grafts can befurther minimized by peritransplant treatments with tolerizing apoptoticdonor cells.

III. Tolerizing Vaccines

Traditionally, vaccines are used to confer immunity to a host. Forexample, injecting an inactivated virus with adjuvant under the skin canlead to temporary or permanent immunity to the active and/or virulentversion of the virus. This can be referred to as a positive vaccine(FIG. 3). However, inactivated cells (e.g., cells from a donor or ananimal genetically different from the donor) that is injectedintravenously, can result in tolerance of a donor cells, or cells withsimilar cellular markers. This can be referred to as a tolerizingvaccine (also referred to as a negative vaccine) (FIG. 3). The inactivecells can be injected without an adjuvant. Alternatively, the inactivecells can be injected with an adjuvant. These tolerizing vaccines can beadvantageous in transplantation, for example, in xenotransplantation, bytolerizing a recipient and preventing rejection. Tolerization can beconferred to a recipient without the use of immunosuppressive therapies.However, in some cases, other immunosuppressive therapies in combinationwith tolerizing vaccines, can decrease transplantation rejection.

FIG. 4 demonstrates an exemplary approach to extending the survival oftransplanted grafts (e.g., xenografts) in a subject (e.g., a human or anon-human primate) with infusion (e.g., intravenous infusion) ofapoptotic cells from the donor for tolerizing vaccination under thecover of transient immunosuppression. A donor can provide xenografts fortransplantation (e.g., islets), as well as cells (e.g., splenocytes) asa tolerizing vaccine. The tolerizing vaccine cells can be apoptoticcells (e.g., by ECDI fixation) and administered to the recipient before(e.g., the first vaccine, on day 7 before the transplantation) and afterthe transplantation (e.g., the booster vaccine, on day 1 after thetransplantation). The tolerizing vaccine can provide transientimmunosuppression that extends the time of survival of the transplantedgrafts (e.g., islets).

Tolerizing vaccines can comprise one or more of the following types ofcells: i) apoptotic cells comprising genotypically identical cells withreduced expression of GGTA1 alone, or GGTA1 and CMAH, or GGTA1, CMAH,and B4GALNT2. This can minimize or eliminate cell-mediated immunity andcell-dependent antibody-mediated immunity to organ, tissue, cell, andcell line grafts (e.g., xenografts) from animals that are genotypicallyidentical with the apoptotic cell vaccine donor animal, or from animalsthat have undergone additional genetic modifications (e.g., suppressionof NLRC5, TAP1, MICA, MICB, CXCL10, C3, CIITA genes or expression oftransgenes comprising two or more polynucleotide inserts of ICP47, CD46,CD55, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5,HLA-G6, or HLA-G7), B2M, CD59, or any functional fragments thereof), butare genotypically similar to the donor animal from which the apoptoticcell vaccine is derived; ii) apoptotic stem cell (e.g., embryonic,pluripotent, placental, induced pluripotent, etc.)-derived donor cells(e.g., leukocytes, lymphocytes, T lymphocytes, B lymphocytes, red bloodcells, graft cells, or any other donor cell) for minimizing oreliminating cell-mediated immunity and cell-dependent antibody-mediatedimmunity to organ, tissue, cell, and cell line grafts (e.g., xenografts)from animals that are genotypically identical with the apoptotic cellvaccine donor animal or from animals that have undergone additionalgenetic modifications (e.g., suppression of NLRC5, TAP1, MICA, MICB,CXCL10, C3, CIITA genes or expression of transgenes comprising two ormore polynucleotide inserts of ICP47, CD46, CD55, HLA-E, HLA-G (e.g.,HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, CD59,or any functional fragments thereof), but are genotypically similar tothe donor animal from which the apoptotic stem cell-derived cell vaccineis derived; iii) apoptotic stem cell (e.g., embryonic, pluripotent,placental, induced pluripotent, etc.)-derived donor cells (leukocytes,lymphocytes, T lymphocytes, B lymphocytes, red blood cells, graft cellssuch as functional islet beta cells, or any other donor cell) forminimizing or eliminating cell-mediated immunity and cell-dependentantibody-mediated immunity to organ, tissue, cell, and cell grafts(e.g., allografts) that are genotypically identical with the human stemcell line or to grafts (e.g., allografts) derived from the same stemcell line that have undergone genetic modifications (e.g., suppressionof NLRC5, TAP1, MICA, MICB, CXCL10, C3, CIITA genes) but are otherwisegenotypically similar to the apoptotic human stem cell-derived donorcell vaccine; iv) apoptotic donor cells, where the cells are madeapoptotic by UV irradiation, gamma-irradiation, or other methods notinvolving incubation in the presence of ECDI. In some cases, tolerizingvaccine cells can be adminstered, e.g., infused (in some casesrepeatedly infused) to a subject in need thereof. Tolerizing vaccinescan be produced by disrupting (e.g., reducing expression) one or moregenes from a cell. For example, genetically modified cells as describedthroughout the application can be used to make a tolerizing vaccine. Forexample, cells can have one or more genes that can be disrupted (e.g.,reduced expression) including glycoprotein galactosyltransferase alpha1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acidhydroxylase-like protein (CMAH), B4GALNT2, and/or any combinationthereof. For example, a cell can have disrupted GGTA1 only, or disruptedCMAH only, or disrupted B4GALNT2 only. A cell can also have disruptedGGTA1 and CMAH, disrupted GGTA1 and B4GALNT2, or disrupted CMAH andB4GALNT2. A cell can have disrupted GGTA1, CMAH, and B4GALNT2. In somecases, the disrupted gene does not include GGTA1. A cell can alsoexpress NLRC5 (endogenously or exogenously), while GGTA1 and/or CMAH aredisrupted. A cell can also have disrupted C3.

A tolerizing vaccine can be produced with cells comprising additionallyexpressing one or more transgenes, e.g., as described throughout theapplication. For example, a tolerizing vaccine can comprise a cellcomprising one or more transgenes comprising one or more polynucleotideinserts of Infected cell protein 47 (ICP47), Cluster of differentiation46 (CD46), Cluster of differentiation 55 (CD55), Cluster ofdifferentiation 59 (CD 59), HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3,HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, PD-L1, PD-L2, CD47, anyfunctional fragments thereof, or any combination thereof. In some cases,a tolerizing vaccine can comprise a genetically modified cell comprisingreduced protein expression of GGTA1, CMAH, and B4GALNT2, and transgenescomprising polynucleotides encoding proteins or functional fragmentsthereof, where the proteins comprise HLA-G1, PD-L1, PD-L2, and CD47. Insome cases, a tolerizing vaccine can comprise a genetically modifiedcell comprising reduced protein expression of GGTA1, CMAH, and B4GALNT2,and transgenes comprising polynucleotides encoding proteins orfunctional fragments thereof, where the proteins comprise HLA-E, PD-L1,PD-L2, and CD47. In some cases, a tolerizing vaccine can comprise a cellcoated with CD47 on its surface. Coating of CD47 on the surface of acell can be accomplished by biotinylating the cell surface followed byincubating these biotinylated cells with a streptavidin-CD47 chimericprotein. For example, a tolerizing vaccine can comprise a cell coatedwith CD47 on its surface, where the cell comprises reduced proteinexpression of GGTA1, CMAH, and B4GALNT2, and transgenes comprisingpolynucleotides encoding proteins or functional fragments thereof, wherethe proteins comprise HLA-G1, PD-L1, and PD-L2. A CD47-coated cell canbe a non-apoptotic cell. Alternative, a CD47 coated cell can be anapoptotic cell.

When administered in a subject, a cell of a tolerizing vaccine can havea circulation half-life. A cell of a tolerizing vaccine can have acirculation half-life of at least or at least about 0.1, 0.5, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 18, 24, 36, 48, 60, or 72 hours. For example,the circulation half-life of the tolerizing vaccine can be from or fromabout 0.1 to 0.5; 0.5 to 1.0; 1.0 to 2.0; 1.0 to 3.0; 1.0 to 4.0; 1.0 to5.0; 5 to 10; 10 to 15; 15 to 24; 24 to 36; 36 to 48; 48 to 60; or 60 to72 hours. A cell in a tolerizing vaccine can be treated to enhance itscirculation half-life. Such treatment can include coating the cell witha protein, e.g., CD47. A cell treated to enhance its circulationhalf-life can be a non-apoptotic cell. A cell treated to enhance itscirculation half-life can be an apoptotic cell. Alternatively, a cell ina tolerizing vaccine can be genetically modified (e.g., insertion of atransgene such as CD47 in its genome) to enhance its circulationhalf-life. A cell genetically modified to enhance its circulationhalf-life can be a non-apoptotic cell. A cell genetically modified toenhance its circulation half-life can be an apoptotic cell.

A tolerizing vaccine can have both one or more disrupted genes (e.g.,reduced expression) and one or more transgenes. Any genes and/ortransgenes as described herein can be used.

A cell that comprises one or more disrupted genes (e.g., reducedexpression) can be used as, or be a part of, a tolerizing vaccine. Inother words, a cell that comprises one or more disrupted genes can be orcan be made into a tolerizing vaccine.

A tolerizing vaccine can have the same genotype and/or phenotype ascells, organs, and/or tissues used in transplantation. Sometimes, thegenotype and/or phenotype of a tolerizing vaccine and a transplant aredifferent. A tolerizing vaccine used for a transplant recipient cancomprise cells from the transplant graft donor. A tolerizing vaccineused for a transplant recipient can comprise cells that are geneticallyand/or phenotypically different from the transplant graft. In somecases, a tolerizing vaccine used for a transplant recipient can comprisecells from the transplant graft donor and cells that are geneticallyand/or phenotypically different from the transplant graft. The cellsthat are genetically and/or phenotypically different from the transplantgraft can be from an animal of the same species of the transplant graftdonor.

A source of cells for a tolerizing vaccine can be from a human ornon-human animal.

Cells as disclosed throughout the application can be made into atolerizing vaccine. For example, a tolerizing vaccine can be made of oneor more transplanted cells disclosed herein. Alternatively, a tolerizingvaccine can be made of one or more cells that are different from any ofthe transplanted cells. For example, the cells made into a tolerizingvaccine can be genotypically and/or phenotypically different from any ofthe transplanted cells. However in some cases, the tolerizing vaccinewill express NLRC5 (endogenously or exogenously). A tolerizing vaccinecan promote survival of cells, organs, and/or tissues intransplantation. A tolerizing vaccine can be derived from non-humananimals that are genotypically identical or similar to donor cells,organs, and/or tissues. For example, a tolerizing vaccine can be cellsderived from pigs (e.g., apoptotic pig cells) that are genotypicallyidentical or similar to donor pig cells, organs, and/or tissues.Subsequently, donor cells, organs, and/or tissues can be used inallografts or xenografts. In some cases, cells for a tolerizing vaccinecan be from genetically modified animals (e.g., pigs) with reducedexpression of GGTA1, CMAH, and B4Ga1NT2, and having transgenes encodingHLA-G (or HLA-E-), human CD47, human PD-L1 and human PD-L2. Graft donoranimals can be generated by further genetically modifying the animals(e.g., pigs) for tolerizing vaccine cells. For example, graft donoranimals can be generated by disrupting additional genes (e.g., NLRC5 (orTAP1), C3, and CXCL10) in the abovementioned animals for tolerizingvaccines cells (FIG. 5).

A tolerizing vaccine can comprise non-human animal cells (e.g.,non-human mammalian cells). For example, non-human animal cells can befrom a pig, a cat, a cattle, a deer, a dog, a ferret, a gaur, a goat, ahorse, a mouse, a mouflon, a mule, a rabbit, a rat, a sheep, or aprimate. Specifically, non-human animal cells can be porcine cells. Atolerizing vaccine can also comprise genetically modified non-humananimal cells. For example, genetically modified non-human animal cellscan be dead cells (e.g., apoptotic cells). A tolerizing vaccine can alsocomprise any genetically modified cells disclosed herein.

Treatment of Cells to Make a Tolerizing Vaccine

A tolerizing vaccine can comprise cells treated with a chemical. In somecases, the treatment can induce apoptosis of the cells. Without beingbound by theory, the apoptotic cells can be picked up by host antigenpresenting cells (e.g., in the spleen) and presented to host immunecells (e.g., T cells) in a non-immunogenic fashion that leads toinduction of anergy in the immune cells (e.g., T cells).

Tolerizing vaccines can comprise apoptotic cells and non-apoptoticcells. An apoptotic cell in a tolerizing vaccine can be geneticallyidentical to a non-apoptotic cell in the tolerizing vaccine.Alternatively, an apoptotic cell in a tolerizing vaccine can begenetically different from a non-apoptotic cell in the tolerizingvaccine. Tolerizing vaccines can comprise fixed cells and non-fixedcells. A fixed cell in a tolerizing vaccine can be geneticallyidentifical to a non-fixed cell in the tolerizing vaccine.Alternatively, a fixed cell in a tolerizing vaccine can be geneticallydifferent from a non-fixed cell in the tolerizing vaccine. In somecases, the fixed cell can be a1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI)-fixed cell.

Cells in a tolerizing vaccine can be fixed using a chemical, e.g., ECDI.The fixation can make the cells apoptotic. A tolerizing vaccine, cells,kits and methods disclosed herein can comprise ECDI and/or ECDItreatment. For example, a tolerizing vaccine can be cells, e.g., thegenetically modified cell as disclosed herein, that are treated with1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). In other words,the genetically modified cells as described throughout can be treatedwith ECDI to create a tolerizing vaccine. A tolerizing vaccine can thenbe used in transplantation to promote survival of cells, organs, and/ortissues that are transplanted. It is also contemplated that ECDIderivatives, functionalized ECDI, and/or substituted ECDI can also beused to treat the cells for a tolerizing vaccine. In some cases, cellsfor a tolerizing vaccine can be treated with any suitable carbodiimidederivatives, e.g., ECDI, N,N′-diisopropylcarbodiimide (DIC),N,N′-dicyclohexylcarbodiimide (DCC), and other carbodiimide derivativesunderstood by those in the art.

Cells for tolerizing vaccines can also be made apoptotic methods notinvolving incubation in the presence of ECDI, e.g., other chemicals orirradiation such as UV irradiation or gamma-irradiation.

ECDI can chemically cross-link free amine and carboxyl groups, and caneffectively induce apoptosis in cells, organs, and/or tissues, e.g.,from animal that gave rise to both a tolerizing vaccine and a donornon-human animal. In other words, the same genetically modified animalcan give rise to a tolerizing vaccine and cells, tissues and/or organsthat are used in transplantation. For example, the genetically modifiedcells as disclosed herein can be treated with ECDI. This ECDI fixationcan lead to the creation of a tolerizing vaccine.

Genetically modified cells that can be used to make a tolerizing vaccinecan be derived from: a spleen (including splenic B cells), liver,peripheral blood (including peripheral blood B cells), lymph nodes,thymus, bone marrow, or any combination thereof. For example, cells canbe spleen cells, e.g., porcine spleen cells. In some cases, cells can beexpanded ex-vivo. In some cases, cells can be derived from fetal,perinatal, neonatal, preweaning, and/or young adult, non-human animals.In some cases, cells can be derived from an embryo of a non-humananimal.

Cells in a tolerizing vaccine can also comprise two or more disrupted(e.g., reduced expression) genes, where the two or more disrupted genescan be glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putativecytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein(CMAH), HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5,HLA-G6, or HLA-G7), B2M, and B4GALNT2, any functional fragments thereof,or any combination thereof. In some cases, the two or more disruptedgenes does not include GGTA1. As described above, disruption can be aknockout or suppression of gene expression. Knockout can be performed bygene editing, for example, by using a CRISPR/cas system. Alternatively,suppression of gene expression can be done by knockdown, for example,using RNA interference, shRNA, one or more dominant negative transgenes.In some cases, cells can further comprise one or more transgenes asdisclosed herein. For example, one or more transgenes can be CD46, CD55,CD59, or any combination thereof.

Cells in a tolerizing vaccine can also be derived from one or more donornon-human animals. In some cases, cells can be derived from the samedonor non-human animal. Cells can be derived from one or more recipientnon-human animals. In some cases, cells can be derived from two or morenon-human animals (e.g., pig).

A tolerizing vaccine can comprise from or from about 0.001 and about5.0, e.g., from or from about 0.001 and 1.0, endotoxin unit per kgbodyweight of a prospective recipient. For example, a tolerizing vaccinecan comprise from or from about 0.01 to 5.0; 0.01 to 4.5; 0.01 to 4.0,0.01 to 3.5; 0.01 to 3.0; 0.01 to 2.5; 0.01 to 2.0; 0.01 to 1.5; 0.01 to1.0; 0.01 to 0.9; 0.01 to 0.8; 0.01 to 0.7; 0.01 to 0.6; 0.01 to 0.5;0.01 to 0.4; 0.01 to 0.3; 0.01 to 0.2; or 0.01 to 0.1 endotoxin unit perkg bodyweight of a prospective recipient.

A tolerizing vaccine can comprise from or from about 1 to 100aggregates, per μl. For example, a tolerizing vaccine can comprise fromor from about 1 to 5; 1 to 10, or 1 to 20 aggregate per μl. A tolerizingvaccine can comprise at least or at least about 1, 5, 10, 20, 50, or 100aggregates.

A tolerizing vaccine can trigger a release from or from about 0.001pg/ml to 10.0 pg/ml, e.g., from or from about 0.001 pg/ml to 1.0 pg/ml,IL-1 beta when about 50,000 frozen to thawed human peripheral bloodmononuclear cells are incubated with about 160,000 cells of thetolerizing vaccine (e.g., pig cells). For example, a tolerizing vaccinetriggers a release of from or from about 0.001 to 10.0; 0.001 to 5.0;0.001 to 1.0; 0.001 to 0.8; 0.001 to 0.2; or 0.001 to 0.1 pg/ml IL-1beta when about 50,000 frozen to thawed human peripheral bloodmononuclear cells are incubated with about 160,000 cell of thetolerizing vaccine (e.g., pig cells). A tolerizing vaccine can trigger arelease of from or from about 0.001 to 2.0 pg/ml, e.g., from or fromabout 0.001 to 0.2 pg/ml, IL-6 when about 50,000 frozen to thawed humanperipheral blood mononuclear cells are incubated with about 160,000cells of the tolerizing vaccine (e.g., pig cells). For example, atolerizing vaccine can trigger a release of from or from about 0.001 to2.0; 0.001 to 1.0; 0.001 to 0.5; or 0.001 to 0.1 pg/ml IL-6 when about50,000 frozen to thawed human peripheral blood mononuclear cells areincubated with about 160,000 cells of the tolerizing vaccine (e.g., pigcells).

A tolerizing vaccine can comprise more than or more than about 60%,e.g., more than or more than about 85%, Annexin V positive, apoptoticcells after a 4 hour or after about 4 hours post-release incubation at37° C. For example, a tolerizing vaccine comprises more than 60%, 70%,80%, 90%, or 99% Annexin V positive, apoptotic cells after about a 4hour post-release incubation at 37° C.

A tolerizing vaccine can include from or from about 0.01% to 10%, e.g.,from or from about 0.01% to 2%, necrotic cells. For example, atolerizing vaccine includes from or from about 0.01% to 10%; 0.01% to7.5%, 0.01% to 5%; 0.01% to 2.5%; or 0.01% to 1% necrotic cells.

Administering a tolerizing vaccine comprising ECDI-treated cells,organs, and/or tissues before, during, and/or after administration ofdonor cells can induce tolerance for cells, organs, and/or tissues in arecipient (e.g., a human or a non-human animal). ECDI-treated cells canbe administered by intravenous infusion.

Tolerance induced by infusion of a tolerizing vaccine comprisingECDI-treated splenocytes is likely dependent on synergistic effectsbetween an intact programmed death 1 receptor—programmed death ligand 1signaling pathway and CD4⁺CD25⁺Foxp3⁺ regulatory T cells.

Cells in a telorizing vaccine can be made into apoptotic cells (e.g.,tolerizing vaccines) not only by ECDI fixation, but also through othermethods. For example, any of the genetically modified cells as disclosedthroughout, e.g., non-human cells animal cells or human cells (includingstem cells), can be made apopototic by exposing the genetically modifiedcells to UV irradiation. The genetically modified cells can also be madeapopototic by exposing it to gamma-irradiation. Other methods, notinvolving ECDI are also comtemplated, for example, by EtOH fixation.

Cells in a tolerizing vaccine, e.g., ECDI-treated cells, antigen-coupledcells, and/or epitope-coupled cells can comprise donor cells (e.g.,cells from the donor of transplant grafts). Cells in a tolerizingvaccine, e.g., ECDI-treated cells, antigen-coupled cells, and/orepitope-coupled cells can comprise recipient cells (e.g., cells from therecipient of transplant grafts). Cells in a tolerizing vaccine, e.g.,ECDI-treated cells, antigen-coupled cells, and/or epitope-coupled cellscan comprise third party (e.g., neither donor nor recipient) cells. Insome cases, third party cells are from a non-human animal of the samespecies as a recipient and/or donor. In other cases, third party cellsare from a non-human animal of a different species as a recipient and/ordonor.

ECDI-treatment of cells can be performed in the presence of one or moreantigens and/or epitopes. ECDI-treated cells can comprise donor,recipient and/or third party cells. Likewise, antigens and/or epitopescan comprise donor, recipient and/or third party antigens and/orepitopes. In some cases, donor cells are coupled to recipient antigensand/or epitopes (e.g., ECDI-induced coupling). For example, solubledonor antigen derived from genetically engineered and genotypicallyidentical donor cells (e.g., porcine cells) is coupled to recipientperipheral blood mononuclear cells with ECDI and the ECDI-coupled cellsare administered via intravenous infusion.

In some cases, recipient cells are coupled to donor antigens and/orepitopes (e.g., ECDI-induced coupling). In some cases, recipient cellsare coupled to third party antigens and/or epitopes (e.g., ECDI-inducedcoupling). In some cases, donor cells are coupled to recipient antigensand/or epitopes (e.g., ECDI-induced coupling). In some cases, donorcells are coupled to third party antigens and/or epitopes (e.g.,ECDI-induced coupling). In some cases, third party cells are coupled todonor antigens and/or epitopes (e.g., ECDI-induced coupling). In somecases, third party cells are coupled to recipient antigens and/orepitopes (e.g., ECDI-induced coupling). For example, soluble donorantigen derived from genetically engineered and genotypically identicaldonor cells (e.g., porcine cells) is coupled to polystyrenenanoparticles with ECDI and the ECDI-coupled cells are administered viaintravenous infusion.

Tolerogenic potency of any of these tolerizing cell vaccines can befurther optimized by coupling to the surface of cells one or more of thefollowing: IFN-g, NF-kB inhibitors (such as curcumin, triptolide,Bay-117085), vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23,or other immunomodulatory molecules that modify the function of hostantigen-presenting cells and host lymphocytes.

These apoptotic cell vaccines can also be complemented by donor cellsengineered to display on their surface molecules (such as FasL, PD-L1,galectin-9, CD8alpha) that trigger apoptotic death of donor-reactivecells.

Tolerizing vaccines dislosed herein can increase the duration ofsurvival of a transplant (e.g., a xenograft or an allograft transplant)in a recipient. Tolerizing vaccines disclosed herein can also reduce oreliminate need for immunosupression following transplantation. Xenograftor allograft transplant can be an organ, tissue, cell or cell line.Xenograft transplants and tolerizing vaccines can also be from differentspecies. Alternatively, xenograft transplants and the tolerizingvaccines can be from the same species. For example, a xenografttransplant and a tolerizing vaccine can be from substantiallygenetically identical individuals (e.g., the same individual).

The ECDI fixed cells can be formulated into a pharmaceuticalcomposition. For example, the ECDI fixed cells can be combined with apharmaceutically acceptable excipient. An excipient that can be used issaline. An excipient that can be used is phosphate buffered saline(PBS). The pharmaceutical compositions can be then used to treatpatients in need of transplantation.

Tolerizing Vaccines Made from Cells Derived Stem Cells

Cells for making tolerizing vaccines can be derived from stem cells.Such cells can include tolerizing apoptotic donor cells that are eitherstem cell-derived functional insulin-secreting islet β cells or othercells differentiated from the identical or genotypically similar stemcell line. These other cells can include leukocytes, lymphocytes, Tlymphocytes, B lymphocytes, red blood cells, or any other donor cell.

These stem-cell derived tolerizing apoptotic donor cells need not begenetically engineered to lack functional expression of MHC class I.Functional expression of MHC class Ion apoptotic donor cells can enhancetheir tolerogenic potential.

Stem cell-derived cells can be made apoptotic by UV irradiation,gamma-irradiation, or other methods not involving incubation in thepresence of ECDI.

These negative cell vaccines can be infused intravenously pretransplantor both pretransplant and at intervals posttransplant, each under thecover of transient immunosuppression including but not limited toantagonistic anti-CD40 antibodies (e.g., humanized 2C10), B celldepleting or targeting antibodies (e.g., rituximab), mTOR inhibitors(e.g., rapamycin), and TNF-alpha inhibitors (e.g., sTNFR, includingetanercept), and IL-6 inhibitors (e.g., anti-IL-6R antibody, includingtocilizumab).

Tolerogenic potency of any of these tolerizing cell vaccines can befurther optimized by coupling to the surface of cells one or more of thefollowing molecules: IFN-g, NF-kB inhibitors (such as curcumin,triptolide, Bay-117085), vitamin D3, siCD40, cobalt protoporphyrin,insulin B9-23, or other immunomodulatory molecules that modify thefunction of host antigen-presenting cells and host lymphocytes.

These apoptotic cell vaccines can also be complemented by donor cellsengineered to display on their surface molecules (such as FasL, PD-L1,galectin-9, CD8alpha) that trigger apoptotic death of donor-reactivecells.

As with human stem cell derived tolerizing vaccines, tolerizingapoptotic donor pig vaccines can be derived from the same cell sources,can express MHC class I antigen, made apoptotic using the same methods,optimized by coupling to the surface of cells one or moreimmunomodulatory molecules, and infused intravenously pretransplant orboth pretransplant and at intervals posttransplant under the cover ofconcomitant immunotherapy.

IV. Method of Making Genetically Modified Non-Human Animals

In order to make a genetically modified non-human animal as describedabove, various techniques can be used. Disclosed herein are a fewexamples to create genetically modified animals. It is to be understoodthat the methods disclosed herein are simply examples, and are not meantto limiting in any way.

Gene Disruption

Gene disruption can be performed by any methods described above, forexample, by knockout, knockdown, RNA interference, dominant negative,etc. A detailed description of the methods are disclosed above in thesection regarding genetically modified non-human animals.

CRISPR/Cas System

Methods described herein can take advantage of a CRISPR/cas system. Forexample, double-strand breaks (DSBs) can be generated using a CRISPR/cassystem, e.g., a type II CRISPR/cas system. A Cas enzyme used in themethods disclosed herein can be Cas9, which catalyzes DNA cleavage.Enzymatic action by Cas9 derived from Streptococcus pyogenes or anyclosely related Cas9 can generate double stranded breaks at target sitesequences which hybridize to 20 nucleotides of a guide sequence and thathave a protospacer-adjacent motif (PAM) following the 20 nucleotides ofthe target sequence.

A vector can be operably linked to an enzyme-coding sequence encoding aCRISPR enzyme, such as a Cas protein. Non-limiting examples of Casproteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t,Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12),Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1,Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3,Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX,Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1,Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, homologues thereof, or modifiedversions thereof. An unmodified CRISPR enzyme can have DNA cleavageactivity, such as Cas9. A CRISPR enzyme can direct cleavage of one orboth strands at a target sequence, such as within a target sequenceand/or within a complement of a target sequence. For example, a CRISPRenzyme can direct cleavage of one or both strands within about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairsfrom the first or last nucleotide of a target sequence. A vector thatencodes a CRISPR enzyme that is mutated to with respect, to acorresponding wild-type enzyme such that the mutated CRISPR enzyme lacksthe ability to cleave one or both strands of a target polynucleotidecontaining a target sequence can be used.

A vector that encodes a CRISPR enzyme comprising one or more nuclearlocalization sequences (NLSs) can be used. For example, there can be orbe about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs used. A CRISPR enzyme cancomprise the NLSs at or near the ammo-terminus, about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs at or near the carboxy-terminus, orany combination of these (e.g., one or more NLS at the ammo-terminus andone or more NLS at the carboxy terminus). When more than one NLS ispresent, each can be selected independently of others, such that asingle NLS can be present in more than one copy and/or in combinationwith one or more other NLSs present in one or more copies.

CRISPR enzymes used in the methods can comprise at most 6 NLSs. An NLSis considered near the N- or C-terminus when the nearest amino acid tothe NLS is within about 50 amino acids along a polypeptide chain fromthe N- or C-terminus, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30,40, or 50 amino acids.

Guide RNA

As used herein, the term “guide RNA” and its grammatical equivalents canrefer to an RNA which can be specific for a target DNA and can form acomplex with Cas protein. An RNA/Cas complex can assist in “guiding” Casprotein to a target DNA.

A method disclosed herein also can comprise introducing into a cell orembryo at least one guide RNA or nucleic acid, e.g., DNA encoding atleast one guide RNA. A guide RNA can interact with a RNA-guidedendonuclease to direct the endonuclease to a specific target site, atwhich site the 5′ end of the guide RNA base pairs with a specificprotospacer sequence in a chromosomal sequence.

A guide RNA can comprise two RNAs, e.g., CRISPR RNA (crRNA) andtransactivating crRNA (tracrRNA). A guide RNA can sometimes comprise asingle-chain RNA, or single guide RNA (sgRNA) formed by fusion of aportion (e.g., a functional portion) of crRNA and tracrRNA. A guide RNAcan also be a dualRNA comprising a crRNA and a tracrRNA. Furthermore, acrRNA can hybridize with a target DNA.

As discussed above, a guide RNA can be an expression product. Forexample, a DNA that encodes a guide RNA can be a vector comprising asequence coding for the guide RNA. A guide RNA can be transferred into acell or organism by transfecting the cell or organism with an isolatedguide RNA or plasmid DNA comprising a sequence coding for the guide RNAand a promoter. A guide RNA can also be transferred into a cell ororganism in other way, such as using virus-mediated gene delivery.

A guide RNA can be isolated. For example, a guide RNA can be transfectedin the form of an isolated RNA into a cell or organism. A guide RNA canbe prepared by in vitro transcription using any in vitro transcriptionsystem known in the art. A guide RNA can be transferred to a cell in theform of isolated RNA rather than in the form of plasmid comprisingencoding sequence for a guide RNA.

A guide RNA can comprise three regions: a first region at the 5′ endthat can be complementary to a target site in a chromosomal sequence, asecond internal region that can form a stem loop structure, and a third3′ region that can be single-stranded. A first region of each guide RNAcan also be different such that each guide RNA guides a fusion proteinto a specific target site. Further, second and third regions of eachguide RNA can be identical in all guide RNAs.

A first region of a guide RNA can be complementary to sequence at atarget site in a chromosomal sequence such that the first region of theguide RNA can base pair with the target site. In some cases, a firstregion of a guide RNA can comprise from or from about 10 nucleotides to25 nucleotides (i.e., from 10 nts to 25 nts; or from about 10 nts toabout 25 nts; or from 10 nts to about 25 nts; or from about 10 nts to 25nts) or more. For example, a region of base pairing between a firstregion of a guide RNA and a target site in a chromosomal sequence can beor can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24,25, or more nucleotides in length. Sometimes, a first region of a guideRNA can be or can be about 19, 20, or 21 nucleotides in length.

A guide RNA can also comprises a second region that forms a secondarystructure. For example, a secondary structure formed by a guide RNA cancomprise a stem (or hairpin) and a loop. A length of a loop and a stemcan vary. For example, a loop can range from or from about 3 to 10nucleotides in length, and a stem can range from or from about 6 to 20base pairs in length. A stem can comprise one or more bulges of 1 to 10or about 10 nucleotides. The overall length of a second region can rangefrom or from about 16 to 60 nucleotides in length. For example, a loopcan be or can be about 4 nucleotides in length and a stem can be or canbe about 12 base pairs.

A guide RNA can also comprise a third region at the 3′ end that can beessentially single-stranded. For example, a third region is sometimesnot complementarity to any chromosomal sequence in a cell of interestand is sometimes not complementarity to the rest of a guide RNA.Further, the length of a third region can vary. A third region can bemore than or more than about 4 nucleotides in length. For example, thelength of a third region can range from or from about 5 to 60nucleotides in length.

A guide RNA can be introduced into a cell or embryo as an RNA molecule.For example, a RNA molecule can be transcribed in vitro and/or can bechemically synthesized. An RNA can be transcribed from a synthetic DNAmolecule, e.g., a gBlocks® gene fragment. A guide RNA can then beintroduced into a cell or embryo as an RNA molecule. A guide RNA canalso be introduced into a cell or embryo in the form of a non-RNAnucleic acid molecule, e.g., DNA molecule. For example, a DNA encoding aguide RNA can be operably linked to promoter control sequence forexpression of the guide RNA in a cell or embryo of interest. A RNAcoding sequence can be operably linked to a promoter sequence that isrecognized by RNA polymerase III (Pol III). Plasmid vectors that can beused to express guide RNA include, but are not limited to, px330 vectorsand px333 vectors. In some cases, a plasmid vector (e.g., px333 vector)can comprise two guide RNA-encoding DNA sequences.

A DNA sequence encoding a guide RNA can also be part of a vector.Further, a vector can comprise additional expression control sequences(e.g., enhancer sequences, Kozak sequences, polyadenylation sequences,transcriptional termination sequences, etc.), selectable markersequences (e.g., antibiotic resistance genes), origins of replication,and the like. A DNA molecule encoding a guide RNA can also be linear. ADNA molecule encoding a guide RNA can also be circular.

When DNA sequences encoding an RNA-guided endonuclease and a guide RNAare introduced into a cell, each DNA sequence can be part of a separatemolecule (e.g., one vector containing an RNA-guided endonuclease codingsequence and a second vector containing a guide RNA coding sequence) orboth can be part of a same molecule (e.g., one vector containing coding(and regulatory) sequence for both an RNA-guided endonuclease and aguide RNA).

Guide RNA can target a gene in a pig or a pig cell. In some cases, guideRNA can target a pig NLRC5 gene, e.g., sequences listed in Table 4. Insome cases, guide RNA can be designed to target pig NLRC5, GGTA1 or CMAHgene. Exemplary oligonucleotides for making the guide RNA are listed inTable 5.

TABLE 4 Exemplary Sequences of the NLRC5 gene to betargeted by guide RNAs SEQ ID No. Sequence (5′-3′) 61ggggaggaagaacttcacct 62 gtaggacgaccctctgtgtg 63 gaccctctgtgtggggtctg 64ggctcggttccattgcaaga 65 gctcggttccattgcaagat 66 ggttccattgcaagatgggc 67gtcccctcctgagtgtcgaa 68 gcctcaggtacagatcaaaa 69 ggacctgggtgccaggaacg 70gtacccagagtcagatcacc 71 gtacccagagtcagatcacc 72 gtgcccttcgacactcagga 73gtgcccttcgacactcagga 74 gtgcccttcgacactcagga 75 gggggccccaaggcagaaga 76ggcagtcttccagtacctgg

TABLE 5 Exemplary oligonucleotides for making guide RNA constructsSEQ ID SEQ Gene No. Forward sequence (5′ to 3′) ID No.Reverse sequence (5′ to 3′) NLRC5 77 acaccggggaggaagaacttcacctg 78aaaacaggtgaagttcttcctccccg NLRC5 79 acaccgtaggacgaccctctgtgtgg 80aaaaccacacagagggtcgtcctacg NLRC5 81 acaccgaccctctgtgtggggtctgg 82aaaaccagaccccacacagagggtcg NLRC5 83 acaccggctcggttccattgcaagag 84aaaactcttgcaatggaaccgagccg NLRC5 85 acaccgctcggttccattgcaagatg 86aaaacatcttgcaatggaaccgagcg NLRC5 87 acaccggttccattgcaagatgggcg 88aaaacgcccatcttgcaatggaaccg NLRC5 89 acaccgtcccctcctgagtgtcgaag 90aaaacttcgacactcaggaggggacg NLRC5 91 acaccgcctcaggtacagatcaaaag 92aaaacttttgatctgtacctgaggcg NLRC5 93 acaccggacctgggtgccaggaacgg 94aaaaccgttcctggcacccaggtccg NLRC5 95 acaccgtacccagagtcagatcaccg 96aaaacggtgatctgactctgggtacg NLRC5 97 acaccgtacccagagtcagatcaccg 98aaaacggtgatctgactctgggtacg NLRC5 99 acaccgtgcccttcgacactcaggag 100aaaactcctgagtgtcgaagggcacg NLRC5 101 acaccgtgcccttcgacactcaggag 102aaaactcctgagtgtcgaagggcacg NLRC5 103 acaccgtgcccttcgacactcaggag 104aaaactcctgagtgtcgaagggcacg NLRC5 105 acaccgggggccccaaggcagaagag 106aaaactcttctgccttggggcccccg NLRC5 107 acaccggcagtcttccagtacctggg 108aaaacccaggtactggaagactgccg GGTA1 109 caccgagaaaataatgaatgtcaa 110aaacttgacattcattattttctc CMAH 111 caccgagtaaggtacgtgatctgt 112aaacacagatcacgtaccttactc

Homologous Recombination

Homologous recombination can also be used for any of the relevantgenetic modifications as disclosed herein. Homologous recombination canpermit site-specific modifications in endogenous genes and thus novelmodifications can be engineered into a genome. For example, the abilityof homologous recombination (gene conversion and classical strandbreakage/rejoining) to transfer genetic sequence information between DNAmolecules can render targeted homologous recombination and can be apowerful method in genetic engineering and gene manipulation.

Cells that have undergone homologous recombination can be identified bya number of methods. For example, a selection method can detect anabsence of an immune response against a cell, for example by a humananti-gal antibody. A selection method can also include assessing a levelof clotting in human blood when exposed to a cell or tissue. Selectionvia antibiotic resistance can be used for screening.

Making Transgenic Non-Human Animals

Random Insertion

One or more transgenes of the methods described herein can be insertedrandomly to any locus in a genome of a cell. These transgenes can befunctional if inserted anywhere in a genome. For instance, a transgenecan encode its own promoter or can be inserted into a position where itis under the control of an endogenous promoter. Alternatively, atransgene can be inserted into a gene, such as an intron of a gene or anexon of a gene, a promoter, or a non-coding region.

A DNA encoding a transgene sequences can be randomly inserted into achromosome of a cell. A random integration can result from any method ofintroducing DNA into a cell known to one of skill in the art. This caninclude, but is not limited to, electroporation, sonoporation, use of agene gun, lipotransfection, calcium phosphate transfection, use ofdendrimers, microinjection, use of viral vectors including adenoviral,AAV, and retroviral vectors, and/or group II ribozymes.

A DNA encoding a transgene can also be designed to include a reportergene so that the presence of the transgene or its expression product canbe detected via activation of the reporter gene. Any reporter gene knownin the art can be used, such as those disclosed above. By selecting incell culture those cells in which a reporter gene has been activated,cells can be selected that contain a transgene.

A DNA encoding a transgene can be introduced into a cell viaelectroporation. A DNA can also be introduced into a cell vialipofection, infection, or transformation. Electroporation and/orlipofection can be used to transfect fibroblast cells.

Expression of a transgene can be verified by an expression assay, forexample, qPCR or by measuring levels of RNA. Expression level can beindicative also of copy number. For example, if expression levels areextremely high, this can indicate that more than one copy of a transgenewas integrated in a genome. Alternatively, high expression can indicatethat a transgene was integrated in a highly transcribed area, forexample, near a highly expressed promoter. Expression can also beverified by measuring protein levels, such as through Western blotting.

Site Specific Insertion

Inserting one or more transgenes in any of the methods disclosed hereincan be site-specific. For example, one or more transgenes can beinserted adjacent to a promoter, for example, adjacent to or near aRosa26 promoter.

Modification of a targeted locus of a cell can be produced byintroducing DNA into cells, where the DNA has homology to the targetlocus. DNA can include a marker gene, allowing for selection of cellscomprising the integrated construct. Homologous DNA in a target vectorcan recombine with a chromosomal DNA at a target locus. A marker genecan be flanked on both sides by homologous DNA sequences, a 3′recombination arm, and a 5′ recombination arm.

A variety of enzymes can catalyze insertion of foreign DNA into a hostgenome. For example, site-specific recombinases can be clustered intotwo protein families with distinct biochemical properties, namelytyrosine recombinases (in which DNA is covalently attached to a tyrosineresidue) and serine recombinases (where covalent attachment occurs at aserine residue). In some cases, recombinases can comprise Cre, fC31integrase (a serine recombinase derived from Streptomyces phage fC31),or bacteriophage derived site-specific recombinases (including Flp,lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4integrase and phage TP901-1 integrase).

Expression control sequences can also be used in constructs. Forexample, an expression control sequence can comprise a constitutivepromoter, which is expressed in a wide variety of cell types. Forexample, among suitable strong constitutive promoters and/or enhancersare expression control sequences from DNA viruses (e.g., SV40, polyomavirus, adenoviruses, adeno-associated virus, pox viruses, CMV, HSV,etc.) or from retroviral LTRs. Tissue-specific promoters can also beused and can be used to direct expression to specific cell lineages.While experiments discussed in the Examples below will be conductedusing a Rosa26 gene promoter, other Rosa26-related promoters capable ofdirecting gene expression can be used to yield similar results, as willbe evident to those of skill in the art. Therefore, the descriptionherein is not meant to be limiting, but rather disclose one of manypossible examples. In some cases, a shorter Rosa26 5′-upstreamsequences, which can nevertheless achieve the same degree of expression,can be used. Also useful are minor DNA sequence variants of a Rosa26promoter, such as point mutations, partial deletions or chemicalmodifications.

A Rosa26 promoter is expressible in mammals. For example, sequences thatare similar to the 5′ flanking sequence of a pig Rosa26 gene, including,but not limited to, promoters of Rosa26 homologues of other species(such as human, cattle, mouse, sheep, goat, rabbit and rat), can also beused. A Rosa26 gene can be sufficiently conserved among differentmammalian species and other mammalian Rosa26 promoters can also be used.

The CRISPR/Cas system can be used to perform site specific insertion.For example, a nick on an insertion site in the genome can be made byCRISPR/cas to facilitate the insertion of a transgene at the insertionsite.

The methods described herein, can utilize techniques which can be usedto allow a DNA or RNA construct entry into a host cell include, but arenot limited to, calcium phosphate/DNA coprecipitation, microinjection ofDNA into a nucleus, electroporation, bacterial protoplast fusion withintact cells, transfection, lipofection, infection, particlebombardment, sperm mediated gene transfer, or any other technique knownby one skilled in the art.

Certain aspects disclosed herein can utilize vectors. Any plasmids andvectors can be used as long as they are replicable and viable in aselected host. Vectors known in the art and those commercially available(and variants or derivatives thereof) can be engineered to include oneor more recombination sites for use in the methods. Vectors that can beused include, but not limited to eukaryotic expression vectors such aspFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen),pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, andpYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia,Inc.), p3′SS, pXT1, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene,Inc.), and pYES2, pAC360, pBlueBa-cHis A, B, and C, pVL1392,pBlueBac111, pCDM8, pcDNA1, pZeoSV, pcDNA3, pREP4, pCEP4, and pEBVHis(Invitrogen, Corp.), and variants or derivatives thereof.

These vectors can be used to express a gene, e.g., a transgene, orportion of a gene of interest. A gene of portion or a gene can beinserted by using known methods, such as restriction enzyme-basedtechniques.

Making Genetically Modified Non-Human Animals Using a Zygote

Making a genetically modified non-human animal using a nucleic acid fromanother genetically modified non-human animal can be done using varioustechniques known in the art, for example, such as by zygotemanipulation.

For example, zygotes can be used to make a similar genetically modifiednon-human animal. A method of making similar genetically modifiednon-human animals comprising a) producing a cell with reduced expressionof one or more genes and/or comprise exogenous polynucleotides disclosedherein, b) generating an embryo using the resulting cell of a); and c)growing the embryo into the genetically modified non-human animal. Thecell of a) can be produced by disrupting (e.g., reducing expression) oneor more genes in the cell (e.g., as described above in a geneticallymodified non-human animals).

This method can be used to make a similar genetically modified non-humananimal disclosed herein. For example, a method of making a geneticallymodified non-human animal can comprise: a) producing a cell with reducedexpression of one or more genes disclosed herein e.g. (as disclosedabove), where the one or more genes comprise NLRC5, TAP1, and/or C3; b)generating an embryo from the resulting cell of a); and c) growing theembryo into the genetically modified non-human animal.

Cells used in this method can be from any disclosed genetically modifiedcells as described herein. For example, disrupted genes are not limitedto NRLC5, TAP1, and/or C3. Other combinations of gene disruptions andtransgenes can be found throughout the disclosure herein. Furthermore, agenetically modified cell can be of any origin, such as from a non-humananimal (as described herein) or genetically modified cells (as describedherein).

A cell of a) in the methods disclosed herein can be a zygote (e.g., acell formed by joining of a sperm and an ovum). A zygote can be formedby joining: i) of a sperm of a wild-type non-human animal and an ovum ofa wild-type non-human animal; ii) a sperm of a wild-type non-humananimal and an ovum of a genetically modified non-human animal; iii) asperm of a genetically modified non-human animal and an ovum of awild-type non-human animal; and/or iv) a sperm of a genetically modifiednon-human animal and an ovum of a genetically modified non-human animal.A non-human animal can be a pig.

One or more genes in a cell of a) in the methods disclosed herein can bedisrupted by generating breaks at desired locations in a genome). Forexample, breaks can be double-stranded breaks (DSBs). DSBs can begenerated using a nuclease comprising Cas (e.g., Cas9), ZFN, TALEN, andmaganuclease. Nuclease can be a naturally-existing or a modifiednuclease. A nucleic acid encoding a nuclease can also be delivered to acell, where the nuclease is expressed.

Following DSBs, one or more genes can be disrupted by DNA repairingmechanisms, such as homologous recombination (HR) and/or nonhomologousend-joining (NHEJ).

A method can comprise inserting one or more transgenes to a genome ofthe cell of a). One or more transgenes can comprise ICP47, CD46, CD55,CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5,HLA-G6, or HLA-G7), B2M, any functional fragments thereof, and/or anycombination thereof.

A method provided herein can comprise inserting one or more transgeneswhere one or more transgenes can be any transgene in any non-humananimal or genetically modified cell disclosed herein. Transgenes can beinserted into a genome of a non-human animal or genetically modifiedcell in a random or targeted manner, as described herein.

Transgenes can also be inserted to a specific locus in a genome of anon-human animal or genetically modified cell, as disclosed herein. Forexample, a transgene can be inserted adjacent to a promoter. A transgenecan be inserted near a promoter that can be at least or at least about1, 10, 50, 100, 500, or 1000 base pairs from a promoter. A gene in somecases and be inserted into a different chromosome and can still becontrol by a promoter. Transgenes can also be inserted at the 3′ regionof the sense strand from a promoter (e.g., downstream of a promoter).Alternatively, transgenes can be inserted at the 5′ region of the sensestrand from a promoter (e.g., upstream of a promoter). Transgenes can beinserted adjacent to a porcine promoter. For example, transgenes can beinserted adjacent to porcine Rosa26 promoter.

A promoter that can be used herein are described throughout theapplication. For example, a promoter that can be used in methods can bea ubiquitous, tissue-specific or an inducible promoter. Expression of atransgene that is inserted adjacent to a promoter can be regulated. Forexample, if a transgene is inserted near or next to a ubiquitouspromoter, the transgene will be expressed in all cells of a non-humananimal. Some ubiquitous promoters can be a CAGGS promoter, an hCMVpromoter, a PGK promoter, an SV40 promoter, or a Rosa26 promoter.

A promoter can be homologous to a promoter sequence present within thegenome of a human or a non-human animal, such as pig, human, cattle,sheep, goat, rabbit, mouse or rat. A promoter can exhibit at least or atleast about 50%, 60%, 70%, 80, 90%, 95%, 96%, 97%, 98%, or 99% homologyto a promoter sequence present within the genome of a human or anon-human animal. A promoter can exhibit 100% homology to a promotersequence present within the genome of a human or a non-human animal. Apromoter can also exhibit at least or at least about 50%, 60%, 70%, 80,90%, 95%, 96%, 97%, 98%, or 99% identity to a promoter sequence presentwithin the genome of a human or a non-human animal. A promoter can alsoexhibit at 100% identity to a promoter sequence present within thegenome of a human or a non-human animal.

Making a Similar Genetically Modified Non-Human Animal Using CellNuclear Transfer

An alternative method of making a genetically modified non-human animalcan be by cell nuclear transfer. A method of making genetically modifiednon-human animals can comprise a) producing a cell with reducedexpression of one or more genes and/or comprise exogenouspolynucleotides disclosed herein; b) providing a second cell andtransferring a nucleus of the resulting cell from a) to the second cellto generate an embryo generating an embryo; c) growing the embryo intothe genetically modified non-human animal. A cell in this method can bean enucleated cell. The cell of a) can be made using any methods, e.g.,gene disruption and/or insertion described herein or known in the art.

This method can be used to make a similar genetically modified non-humananimal disclosed herein. For example, a method of making a geneticallymodified non-human animal can comprise: a) producing a cell with reducedexpression of NLRC5, TAP1 and/or C3; b) providing a second cell andtransferring a nucleus of the resulting cell from a) to the second cellto generate an embryo; and c) growing the embryo to the geneticallymodified non-human animal. A cell in this method can be an enucleatedcell.

Cells used in this method can be from any disclosed genetically modifiedcells as described herein. For example, disrupted genes are not limitedto NRLC5, TAP1, and/or C3. Other combinations of gene disruptions andtransgenes can be found throughout disclosure herein. For example, amethod can comprise providing a first cell from any non-human animaldisclosed herein; providing a second cell; transferring a nucleus of thefirst cell of a) to the second cell of b); generating an embryo from theproduct of c); and growing the embryo to the genetically modifiednon-human animal.

A cell of a) in the methods disclosed herein can be a zygote. The zygotecan be formed by joining: i) of a sperm of a wild-type non-human animaland an ovum of a wild-type non-human animal; ii) a sperm of a wild-typenon-human animal and an ovum of a genetically modified non-human animal;iii) a sperm of a genetically modified non-human animal and an ovum of awild-type non-human animal; and/or iv) a sperm of a genetically modifiednon-human animal and an ovum of a genetically modified non-human animal.A non-human animal can be a pig.

One or more genes in a cell of a) in the methods disclosed herein can bedisrupted by generating breaks at desired locations in the genome. Forexample, breaks can be double-stranded breaks (DSBs). DSBs can begenerated using a nuclease comprising Cas (e.g., Cas9), ZFN, TALEN, andmaganuclease. Nuclease can be a naturally-existing or a modifiednuclease. A nucleic acid encoding a nuclease can be delivered to a cell,where the nuclease is expressed. Cas9 and guide RNA targeting a gene ina cell can be delivered to the cell. In some cases, mRNA moleculesencoding Cas9 and guide RNA can be injected into a cell. In some cases,a plasmid encoding Cas9 and a different plasmid encoding guide RNA canbe delivered into a cell (e.g., by infection). In some cases, a plasmidencoding both Cas9 and guide RNA can be delivered into a cell (e.g., byinfection).

As described above, following DSBs, one or more genes can be disruptedby DNA repairing mechanisms, such as homologous recombination (HR)and/or nonhomologous end-joining (NHEJ). A method can comprise insertingone or more transgenes to a genome of the cell of a). One or moretransgenes can comprise ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g.,HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, anyfunctional fragments thereof, and/or any combination thereof.

The methods provided herein can comprise inserting one or moretransgenes where the one or more transgenes can be any transgene in anynon-human animal or genetically modified cell disclosed herein.

Also disclosed herein are methods of making a non-human animal using acell from a genetically modified non-human animal. A cell can be fromany genetically modified non-human animal disclosed herein. A method cancomprise: a) providing a cell from a genetically identified non-humananimal; b) providing a cell; c) transferring a nucleus of the cell of a)to the cell of b); c) generating an embryo from the product of c); andd) growing the embryo to the genetically modified non-human animal. Acell of this method can be an enucleated cell.

Further, cells of a) in the methods can be any cell from a geneticallymodified non-human animal. For example, a cell of a) in methodsdisclosed herein can be a somatic cell, such as a fibroblast cell or afetal fibroblast cell.

An enucleated cell in the methods can be any cell from an organism. Forexample, an enucleated cell is a porcine cell. An enucleated cell can bean ovum, for example, an enucleated unfertilized ovum.

Genetically modified non-human animal disclosed herein can be made usingany suitable techniques known in the art. For example, these techniquesinclude, but are not limited to, microinjection (e.g., of pronuclei),sperm-mediated gene transfer, electroporation of ova or zygotes, and/ornuclear transplantation.

A method of making similar genetically modified non-human animals cancomprise a) disrupting one or more genes in a cell, b) generating anembryo using the resulting cell of a); and c) growing the embryo intothe genetically modified non-human animal.

A cell of a) in the methods disclosed herein can be a somatic cell.There is no limitation on a type or source of a somatic cell. Forexample, it can be from a pig or from cultured cell lines or any otherviable cell. A cell can also be a dermal cell, a nerve cell, a cumuluscell, an oviduct epithelial cell, a fibroblast cell (e.g., a fetalfibroblast cell), or hepatocyte. A cell of a) in the methods disclosedherein can be from a wild-type non-human animal, a genetically modifiednon-human animal, or a genetically modified cell. Furthermore, a cell ofb) can be an enucleated ovum (e.g., an enucleated unfertilized ovum).

Enucleation can also be performed by known methods. For example,metaphase II oocytes can be placed in either HECM, optionally containingor containing about 7-10 micrograms per milliliter cytochalasin B, forimmediate enucleation, or can be placed in a suitable medium (e.g., anembryo culture medium such as CRlaa, plus 10% estrus cow serum), andthen enucleated later (e.g., not more than 24 hours later or 16-18 hourslater). Enucleation can also be accomplished microsurgically using amicropipette to remove the polar body and the adjacent cytoplasm.Oocytes can then be screened to identify those of which have beensuccessfully enucleated. One way to screen oocytes can be to stain theoocytes with or with about 3-10 microgram per milliliter 33342 Hoechstdye in suitable holding medium, and then view the oocytes underultraviolet irradiation for less than 10 seconds. Oocytes that have beensuccessfully enucleated can then be placed in a suitable culture medium,for example, CRlaa plus 10% serum. The handling of oocytes can also beoptimized for nuclear transfer.

The embryos generated herein can be transferred to surrogate non-humananimals (e.g., pigs) to produce offspring (e.g., piglets). For example,the embryos can be transferred to the oviduct of recipient gilts on theday or 1 day after estrus e.g., following mid-line laparotomy undergeneral anesthesia. Pregnancy can be diagnosed, e.g., by ultrasound.Pregnancy can be diagnosed after or after about 28 days from thetransfer. The pregnancy can then checked at or at about 2-week intervalsby ultrasound examination. All of the microinjected offspring (e.g.,piglets) can be delivered by natural birth. Information of the pregnancyand delivery (e.g., time of pregnancy, rates of pregnancy, number ofoffspring, survival rate, etc.) can be documented. The genotypes andphenotypes of the offspring can be measured using any methods describedthrough the application such as sequencing (e.g., next-generationsequencing).

Cultured cells can be used immediately for nuclear transfer (e.g.,somatic cell nuclear transfer), embryo transfer, and/or inducingpregnancy, allowing embryos derived from healthy stable geneticmodifications give rise to offspring (e.g., piglets). Such approach canreduce time and cost, e.g., months of costly cell screening that mayresult in genetically modified cells fail to produce healthy piglets.

Embryo growing and transferring can be performed using standardprocedures used in the embryo growing and transfer industry. Forexample, surrogate mothers can be used. Embryos can also be grown andtransferred in culture, for example, by using incubators. In some cases,an embryo can be transferred to an animal, e.g., a surrogate animal, toestablish a pregnancy.

It can be desirable to replicate or generate a plurality of geneticallymodified non-human animals that have identical genotypes and/orphenotypes disclosed herein. For example, a genetically modifiednon-human animal can be replicated by breeding (e.g., selectivebreading). A genetically modified non-human animal can be replicated bynuclear transfer (e.g., somatic cell nuclear transfer) or introductionof DNA into a cell (e.g., oocytes, sperm, zygotes or embryonic stemcells). These methods can be reproduced a plurality of times toreplicate or generate a plurality of a genetically modified non-humananimal disclosed herein. In some cases, cells can be isolated from thefetuses of a pregnant genetically modified non-human animal. Theisolated cells (e.g., fetal cells) can be used for generating aplurality of genetically modified non-human animals similar or identicalto the pregnant animal. For example, the isolated fetal cells canprovide donor nuclei for generating genetically modified animals bynuclear transfer, (e.g., somatic cell nuclear transfer).

V. Methods of Use

Cells, organs, and/or tissues can be extracted from a non-human animalas described herein. Cells, organs, and/or tissues can be geneticallyaltered ex vivo and used accordingly. These cells, organs, and/ortissues can be used for cell-based therapies. These cells, organs,and/or tissues can be used to treat or prevent disease in a recipient(e.g., a human or non-human animal). Surprisingly, the geneticmodifications as described herein can help prevent rejection.Additionally, cells, organs, and/or tissues can be made into tolerizingvaccines to also help tolerize the immune system to transplantation.Further, tolerizing vaccines can temper the immune system, including,abrogating autoimmune responses.

Disclosed herein are methods for treating a disease in a subject in needthereof can comprise administering a tolerizing vaccine to the subject;administering a pharmaceutical agent that inhibits T cell activation tothe subject; and transplanting a genetically modified cell to thesubject. The pharmaceutical agent that inhibits T cell activation can bean antibody. The antibody can be an anti-CD40 antibody disclosed herein.The cell transplanted to the subject can be any genetically modifiedcell described throughout the application. The tissue or organtransplanted to the subject can comprise one or more of the geneticallymodified cells. In some cases, the methods can further compriseadministering one or more immunosuppression agent described in theapplication, such as further comprising providing to the recipient oneor more of a B-cell depleting antibody, an mTOR inhibitor, a TNF-alphainhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent (e.g.,cyclophosphamide), and a complement C3 or C5 inhibitor.

Also disclosed herein are methods for treating a disease, comprisingtransplanting one or more cells to a subject in need thereof. The one ormore cells can be any genetically modified cells disclosed herein. Insome cases, the methods can comprise transplanting a tissue or organcomprising the one or more cells (e.g., genetically modified cells) tothe subject in need thereof.

Described herein are methods of treating or preventing a disease in arecipient (e.g., a human or non-human animal) comprising transplantingto the recipient (e.g., a human or non-human animal) one or more cells(including organs and/or tissues) derived from a genetically modifiednon-human animal comprising one or more genes with reduced expression.One or more cells can be derived from a genetically modified non-humananimal as described throughout.

The methods disclosed herein can be used for treating or preventingdisease including, but not limited to, diabetes, cardiovasculardiseases, lung diseases, liver diseases, skin diseases, or neurologicaldisorders. For example, the methods can be used for treating orpreventing Parkinson's disease or Alzheimer's disease. The methods canalso be used for treating or preventing diabetes, including type 1, type2, cystic fibrosis related, surgical diabetes, gestational diabetes,mitochondrial diabetes, or combination thereof. In some cases, themethods can be used for treating or preventing hereditary diabetes or aform of hereditary diabetes. Further, the methods can be used fortreating or preventing type 1 diabetes. The methods can also be used fortreating or preventing type 2 diabetes. The methods can be used fortreating or preventing pre-diabetes.

For example, when treating diabetes, genetically modified splenocytescan be fixed with ECDI and given to a recipient. Further, geneticallymodified pancreatic islet cells can be grafted into the same recipientto produce insulin. Genetically modified splenocytes and pancreaticislet cells can be genetically identical and can also be derived fromthe same genetically modified non-human animal.

Provided herein include i) genetically modified cells, tissues or organsfor use in administering to a subject in need thereof to treat acondition in the subject; ii) a tolerizing vaccine for use inimmunotolerizing the subject to a graft, where the tolerizing vaccinecomprise a genetically modified cell, tissue, or organ; iii) one or morepharmaceutical agents for use in inhibiting T cell activation, B cellactivation, dendritic cell activation, or a combination thereof in thesubject; or iv) any combination thereof.

Also provided herein include genetically modified cells, tissues ororgans for use in administering to a subject in need thereof to treat acondition in the subject. The subject can have been or become tolerizedto the genetically modified cell, tissue or organ by use of a tolerizingvaccine. Further, the subject can be administered one or morepharmaceutical agents that inhibit T cell activation, B cell activation,dendritic cell activation, or a combination thereof.

Transplantation

The methods disclosed herein can comprise transplanting. Transplantingcan be autotransplanting, allotransplanting, xenotransplanting, or anyother transplanting. For example, transplanting can bexenotransplanting. Transplanting can also be allotransplanting.

“Xenotransplantation” and its grammatical equivalents as used herein canencompass any procedure that involves transplantation, implantation, orinfusion of cells, tissues, or organs into a recipient, where therecipient and donor are different species. Transplantation of the cells,organs, and/or tissues described herein can be used forxenotransplantation in into humans. Xenotransplantation includes but isnot limited to vascularized xenotransplant, partially vascularizedxenotransplant, unvascularized xenotransplant, xenodressings,xenobandages, and xenostructures.

“Allotransplantation” and its grammatical equivalents as used herein canencompasses any procedure that involves transplantation, implantation,or infusion of cells, tissues, or organs into a recipient, where therecipient and donor are the same species. Transplantation of the cells,organs, and/or tissues described herein can be used forallotransplantation in into humans. Allotransplantation includes but isnot limited to vascularized allotransplant, partially vascularizedallotransplant, unvascularized allotransplant, allodressings,allobandages, and allostructures.

After treatment (e.g., any of the treatment as disclosed herein),transplant rejection can be improved as compared to when one or morewild-type cells is transplanted into a recipient. For example,transplant rejection can be hyperacute rejection. Transplant rejectioncan also be acute rejection. Other types of rejection can includechronic rejection. Transplant rejection can also be cell-mediatedrejection or T cell-mediated rejection. Transplant rejection can also benatural killer cell-mediated rejection.

“Improving” and its grammatical equivalents as used herein can mean anyimprovement recognized by one of skill in the art. For example,improving transplantation can mean lessening hyperacute rejection, whichcan encompass a decrease, lessening, or diminishing of an undesirableeffect or symptom.

The disclosure describes methods of treatment or preventing diabetes orprediabetes. For example, the methods include but are not limited to,administering one or more pancreatic islet cell(s) from a donornon-human animal described herein to a recipient, or a recipient in needthereof. The methods can be transplantation or, in some cases,xenotransplantation. The donor animal can be a non-human animal. Arecipient can be a primate, for example, a non-human primate including,but not limited to, a monkey. A recipient can be a human and in somecases, a human with diabetes or pre-diabetes. In some cases, whether apatient with diabetes or pre-diabetes can be treated withtransplantation can be determined using an algorithm, e.g., as describedin Diabetes Care 2015; 38:1016-1029, which is incorporated herein byreference in its entirety.

The methods can also include methods of xenotransplantation where thetransgenic cells, tissues and/or organs, e.g., pancreatic tissues orcells, provided herein are transplanted into a primate, e.g., a human,and, after transplant, the primate requires less or no immunosuppressivetherapy. Less or no immunosuppressive therapy includes, but is notlimited to, a reduction (or complete elimination of) in dose of theimmunosuppressive drug(s)/agent(s) compared to that required by othermethods; a reduction (or complete elimination of) in the number of typesof immunosuppressive drug(s)/agent(s) compared to that required by othermethods; a reduction (or complete elimination of) in the duration ofimmunosuppression treatment compared to that required by other methods;and/or a reduction (or complete elimination of) in maintenanceimmunosuppression compared to that required by other methods.

The methods disclosed herein can be used for treating or preventingdisease in a recipient (e.g., a human or non-human animal). A recipientcan be any non-human animal or a human. For example, a recipient can bea mammal. Other examples of recipient include but are not limited toprimates, e.g., a monkey, a chimpanzee, a bamboo, or a human. If arecipient is a human, the recipient can be a human in need thereof. Themethods described herein can also be used in non-primate, non-humanrecipients, for example, a recipient can be a pet animal, including, butnot limited to, a dog, a cat, a horse, a wolf, a rabbit, a ferret, agerbil, a hamster, a chinchilla, a fancy rat, a guinea pig, a canary, aparakeet, or a parrot. If a recipient is a pet animal, the pet animalcan be in need thereof. For example, a recipient can be a dog in needthereof or a cat in need thereof.

Transplanting can be by any transplanting known to the art. Graft can betransplanted to various sites in a recipient. Sites can include, but notlimited to, liver subcapsular space, splenic subcapsular space, renalsubcapsular space, omentum, gastric or intestinal submucosa, vascularsegment of small intestine, venous sac, testis, brain, spleen, orcornea. For example, transplanting can be subcapsular transplanting.Transplanting can also be intramuscular transplanting. Transplanting canbe intraportal transplanting.

Transplanting can be of one or more cells, tissues, and/or organs from ahuman or non-human animal. For example, the tissue and/or organs can be,or the one or more cells can be from, a brain, heart, lungs, eye,stomach, pancreas, kidneys, liver, intestines, uterus, bladder, skin,hair, nails, ears, glands, nose, mouth, lips, spleen, gums, teeth,tongue, salivary glands, tonsils, pharynx, esophagus, large intestine,small intestine, rectum, anus, thyroid gland, thymus gland, bones,cartilage, tendons, ligaments, suprarenal capsule, skeletal muscles,smooth muscles, blood vessels, blood, spinal cord, trachea, ureters,urethra, hypothalamus, pituitary, pylorus, adrenal glands, ovaries,oviducts, uterus, vagina, mammary glands, testes, seminal vesicles,penis, lymph, lymph nodes or lymph vessels. The one or more cells canalso be from a brain, heart, liver, skin, intestine, lung, kidney, eye,small bowel, or pancreas. The one or more cells are from a pancreas,kidney, eye, liver, small bowel, lung, or heart. The one or more cellscan be from a pancreas. The one or more cells can be pancreatic isletcells, for example, pancreatic β cells. Further, the one or more cellscan be pancreatic islet cells and/or cell clusters or the like,including, but not limited to pancreatic α cells, pancreatic β cells,pancreatic δ cells, pancreatic F cells (e.g., PP cells), or pancreatic εcells. In one instance, the one or more cells can be pancreatic α cells.In another instance, the one or more cells can be pancreatic β cells.

As discussed above, a genetically modified non-human animal can be usedin xenograft (e.g., cells, tissues and/or organ) donation. Solely forillustrative purposes, genetically modified non-human animals, e.g.,pigs, can be used as donors of pancreatic tissue, including but notlimited to, pancreatic islets and/or islet cells. Pancreatic tissue orcells derived from such tissue can comprise pancreatic islet cells, orislets, or islet-cell clusters. For example, cells can be pancreaticislets which can be transplanted. More specifically, cells can bepancreatic β cells. Cells also can be insulin-producing. Alternatively,cells can be islet-like cells. Islet cell clusters can include any oneor more of α, β, δ, PP or ε cells. Aptly the disease to be treated bymethods and compositions herein can be diabetes. Aptly thetransplantable grafts can be pancreatic islets and/or cells frompancreatic islets. Aptly the modification to the transgenic animal is tothe pancreatic islets or cells from the pancreatic islets. Aptly thepancreatic islets or cells from the pancreatic islets are porcine. Insome cases, cells from the pancreatic islets are or include pancreatic βcells.

Donor non-human animals can be at any stage of development including,but not limited to, fetal, neonatal, young and adult. For example, donorcells islet cells can be isolated from adult non-human animals. Donorcells, e.g., islet cells, can also be isolated from fetal or neonatalnon-human animals. Donor non-human animals can be under the age of 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 year(s). For example, islet cells can beisolated from a non-human animal under the age of 6 years. Islet cellscan also be isolated from a non-human animal under the age of 3 years.Donors can be non-human animals and can be any age from or from about 0(including a fetus) to 2; 2 to 4; 4 to 6; 6 to 8; or 8 to 10 years. Anon-human animal can be older than or than about 10 years. Donor cellscan be from a human as well.

Islet cells can be isolated from non-human animals of varying ages. Forexample, islet cells can be isolated from or from about newborn to 2year old non-human animals. Islets cells can also be isolated from orfrom about fetal to 2 year old non-human animals. Islets cells can beisolated from or from about 6 months old to 2 year old non-humananimals. Islets cells can also be isolated from or from about 7 monthsold to 1 year old non-human animals. Islets cells can be isolated fromor from about 2-3 year old non-human animals. In some cases, non-humananimals can be less than 0 years (e.g., a fetus or embryo). In somecases, neonatal islets can be more hearty and consistent post-isolationthan adult islets, can be more resistant to oxidative stress, canexhibit significant growth potential (likely from a nascent islet stemcell subpopulation), such that they can have the ability to proliferatepost-transplantation and engraftment in a transplantation site.

With regards to treating diabetes, neonatal islets can have thedisadvantage that it can take them up to or up to about 4-6 weeks tomature enough such that they produce significant levels of insulin, butthis can be overcome by treatment with exogenous insulin for a periodsufficient for the maturation of the neonatal islets. In xenografttransplantation, survival and functional engraftment of neo-natal isletscan be determined by measuring donor-specific c-peptide levels, whichare easily distinguished from any recipient endogenous c-peptide.

As discussed above, adult cells can be isolated. For example, adultnon-human animal islets, e.g., adult porcine cells, can be isolated.Islets can then be cultured for or for about 1-3 days prior totransplantation in order to deplete the preparation of contaminatingexocrine tissue. Prior to treatment, islets can be counted, andviability assessed by double fluorescent calcein-AM and propidium iodidestaining. Islet cell viability >75% can be used. However, cell viabilitygreater than or greater than about 40%, 50%, 60%, 70%, 80%, 90%, 95%,99% can be used. For example, cells that exhibit a viability from orfrom about 40% to 50%; 50% to 60%; 60% to 70%; 70% to 80%; 80% to 90%;90% to 95%, or 90% to 100% can be used. Additionally, purity can begreater than or greater than about 80% islets/whole tissue. Purity canalso be at least or at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or99% islets/whole tissue. For example, purity can be from or can be fromabout 40% to 50%; 50% to 60%; 60% to 70%; 70% to 80%; 80% to 90%; 90% to100%; 90% to 95%, or 95% to 100%.

Functional properties of islets, including dynamic perifusion andviability, can be determined in vitro prior to treatment (Balamurugan,2006). For example, non-human animal islet cells, e.g., transgenicporcine islet cells can be cultured in vitro to expand, mature, and/orpurify them so that they are suitable for grafting.

Islet cells can also be isolated by standard collagenase digestion ofminced pancreas. For example, using aseptic techniques, glands can bedistended with tissue dissociating enzymes (a mixture of purifiedenzymes formulated for rapid dissociation of a pancreas and maximalrecovery of healthy, intact, and functional islets of Langerhans, wheretarget substrates for these enzymes are not fully identified, but arepresumed to be collagen and non-collagen proteins, which compriseintercellular matrix of pancreatic acinar tissue) (1.5 mg/ml), trimmedof excess fat, blood vessels and connective tissue, minced, and digestedat 37 degree C. in a shaking water bath for 15 minutes at 120 rpm.Digestion can be achieved using lignocaine mixed with tissuedissociating enzymes to avoid cell damage during digestion. Followingdigestion, the cells can be passed through a sterile 50 mm to 1000 mmmesh, e.g., 100 mm, 200 mm, 300 mm, 400 mm, 500 mm, 600 mm, 700 mm, 800mm, 900 mm, or 1000 mm mesh into a sterile beaker. Additionally, asecond digestion process can be used for any undigested tissue.

Islets can also be isolated from the adult pig pancreas (Brandhorst etal., 1999). The pancreas is retrieved from a suitable source pig,peri-pancreatic tissue is removed, the pancreas is divided into thesplenic lobe and in the duodenal/connecting lobe, the ducts of eachlobes are cannulated, and the lobes are distended with tissuedissociating enzymes. The pancreatic lobes are placed into a Ricordichamber, the temperature is gradually increased to 28 to 32° C., and thepancreatic lobes are dissociated by means of enzymatic activity andmechanical forces. Liberated islets are separated from acinar and ductaltissue using continuous density gradients. Purified pancreatic isletsare cultured for or for about 2 to 7 days, subjected tocharacterization, and islet products meeting all specifications arereleased for transplantation (Korbutt et al., 2009).

Donor cells, organs, and/or tissues before, after, and/or duringtransplantation can be functional. For example, transplanted cells,organs, and/or tissues can be functional for at least or at least about1, 5, 10, 20, 30 days after transplantation. Transplanted cells, organs,and/or tissues can be functional for at least or at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 months after transplantation.Transplanted cells, organs, and/or tissues can be functional for atleast or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30years after transplantation. In some cases, transplanted cells, organs,and/or tissues can be functional for up to the lifetime of a recipient.This can indicate that transplantation was successful. This can alsoindicate that there is no rejection of the transplanted cells, tissues,and/or organs.

Further, transplanted cells, organs, and/or tissues can function at 100%of its normal intended operation. Transplanted cells, organs, and/ortissues can also function at least or at least about 50, 60, 65, 70, 75,80, 85, 90, 95, 99, or 100% of its normal intended operation, e.g., fromor from about 50 to 60; 60 to 70; 70 to 80; 80 to 90; 90 to 100%. Incertain instances, the transplanted cells, organs, and/or tissues canfunction at greater 100% of its normal intended operation (when comparedto a normal functioning non-transplanted cell, organ, or tissue asdetermined by the American Medical Association). For example, thetransplanted cells, organs, and/or tissues can function at or at about110, 120, 130, 140, 150, 175, 200% or greater of its normal intendedoperation, e.g., from or from about 100 to 125; 125 to 150; 150 to 175;175 to 200%.

In certain instances, transplanted cells can be functional for at leastor at least about 1 day. Transplanted cells can also functional for atleast or at least about 7 day. Transplanted cells can be functional forat least or at least about 14 day. Transplanted cells can be functionalfor at least or at least about 21 day. Transplanted cells can befunctional for at least or at least about 28 day. Transplanted cells canbe functional for at least or at least about 60 days.

Another indication of successful transplantation can be the days arecipient does not require immunosuppressive therapy. For example, aftertreatment (e.g., transplantation) provided herein, a recipient canrequire no immunosuppressive therapy for at least or at least about 1,5, 10, 100, 365, 500, 800, 1000, 2000, 4000 or more days. This canindicate that transplantation was successful. This can also indicatethat there is no rejection of the transplanted cells, tissues, and/ororgans.

In some cases, a recipient can require no immunosuppressive therapy forat least or at least about 1 day. A recipient can also require noimmunosuppressive therapy for at least or at least about 7 days. Arecipient can require no immunosuppressive therapy for at least or atleast about 14 days. A recipient can require no immunosuppressivetherapy for at least or at least about 21 days. A recipient can requireno immunosuppressive therapy for at least or at least about 28 days. Arecipient can require no immunosuppressive therapy for at least or atleast about 60 days. Furthermore, a recipient can require noimmunosuppressive therapy for at least or at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 years, e.g., for at least or atleast about 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to 15;15 to 20; 20 to 25; 25 to 50 years.

Another indication of successful transplantation can be the days arecipient requires reduced immunosuppressive therapy. For example, afterthe treatment provided herein, a recipient can require reducedimmunosuppressive therapy for at least or at least about 1, 5, 10, 50,100, 200, 300, 365, 400, 500 days, e.g., for at least or at least about1 to 30; 30 to 120; 120 to 365; 365 to 500 days. This can indicate thattransplantation was successful. This can also indicate that there is noor minimal rejection of the transplanted cells, tissues, and/or organs.

For example, a recipient can require reduced immunosuppressive therapyfor at least or at least about 1 day. A recipient can also requirereduced immunosuppressive therapy for at least 7 days. A recipient canrequire reduced immunosuppressive therapy for at least or at least about14 days. A recipient can require reduced immunosuppressive therapy forat least or at least about 21 days. A recipient can require reducedimmunosuppressive therapy for at least or at least about 28 days. Arecipient can require reduced immunosuppressive therapy for at least orat least about 60 days. Furthermore, a recipient can require reducedimmunosuppressive therapy for at least or at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 years, e.g., for at least or atleast about 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to 15;15 to 20; 20 to 25; 25 to 50 years.

“Reduced” and its grammatical equivalents as used herein can refer toless immunosuppressive therapy compared to a required immunosuppressivetherapy when one or more wild-type cells is transplanted into arecipient.

Immunosuppressive Therapy

Immunosuppressive therapy can comprise any treatment that suppresses theimmune system. Immunosuppressive therapy can help to alleviate,minimize, or eliminate transplant rejection in a recipient. For example,immunosuppressive therapy can comprise immuno-suppressive drugs.Immunosuppressive drugs that can be used before, during and/or aftertransplant are any known to one of skill in the art and include, but arenot limited to, MMF (mycophenolate mofetil (Cellcept)), ATG(anti-thymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10,ASKP1240, CCFZ533X2201), alemtuzumab (Campath), anti-CD20 (rituximab),anti-IL-6R antibody (tocilizumab, Actemra), anti-IL-6 antibody(sarilumab, olokizumab), CTLA4-Ig (Abatacept/Orencia), belatacept(LEA29Y), sirolimus (Rapimune), everolimus, tacrolimus (Prograf),daclizumab (Ze-napax), basiliximab (Simulect), infliximab (Remicade),cyclosporin, deoxyspergualin, soluble complement receptor 1, cobra venomfactor, compstatin, anti C5 antibody (eculizumab/Soliris),methylprednisolone, FTY720, everolimus, leflunomide, anti-IL-2R-Ab,rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody,and anti-CD122 antibody. Furthermore, one or more than oneimmunosuppressive agents/drugs can be used together or sequentially. Oneor more than one immunosuppressive agents/drugs can be used forinduction therapy or for maintenance therapy. The same or differentdrugs can be used during induction and maintenance stages. In somecases, daclizumab (Zenapax) can be used for induction therapy andtacrolimus (Prograf) and sirolimus (Rapimune) can be used formaintenance therapy. Daclizumab (Zenapax) can also be used for inductiontherapy and low dose tacrolimus (Prograf) and low dose sirolimus(Rapimune) can be used for maintenance therapy. Immunosuppression canalso be achieved using non-drug regimens including, but not limited to,whole body irradiation, thymic irradiation, and full and/or partialsplenectomy. These techniques can also be used in combination with oneor more immuno-suppressive drug.

Transgenic pancreatic islet cells can be transplanted using any meansknown in the art, including, but not limited to, introduction via arecipient organism's portal vein, liver subcapsular space, splenicsubcapsular space, renal subcapsular space, omentum, gastric orintestinal submucosa, vascular segment of small intestine, venous sac,testis, brain, cornea or spleen. For example, a method ofxenotransplantation can be to transplant pancreatic cells, e.g., porcinepancreatic cells, provided herein into a primate, e.g., a human, whereislets are administered by intraportal infusion. A method ofxenotransplantation can be provided to transplant pancreatic cellsprovided herein into a primate where islets are administered via theintraperitoneal space, renal subcapsule, renal capsule, omentum, or viapancreatic bed infusion. For example, transplanting can be subcapsulartransplanting, intramuscular transplanting, or intraportaltransplanting.

Both allotransplants and xenotransplants can sometimes be subject torecurrent autoimmunity. For example, with regards to islet celltransplantation, islet β cells can be attacked and destroyed aftertransplantation by autoreactive T cells, for example, by CD8+autoreactive T cells, and autoreactive antibodies. When recipients aregiven tolerizing vaccines autoimmune recurrence can be prevented. Forexample, when tolerizing vaccines are engineered to also presentautoantigens such as insulin B9-23 on the surface of apoptotic carriercells or microparticles such as polystyrene particles, tolerance toautoantigens can be restored, and autoimmune recurrence can beprevented. For example, with respect to diabetes, the tolerizing vaccineas disclosed herein can prevent the onset of autoimmune Type 1 diabetesor prevent autoimmune recurrence in transplanted islet β cells.

The tolerizing vaccine can also be given to a recipient to prevent ortreat diabetes (e.g., type 1, type 2, gestational, surgical, cysticfibrosis-related diabetes, or mitochondrial diabetes. In some cases, adisease can be hereditary diabetes or a type of hereditary diabetes).

Additionally, for both allotransplants and xenotransplants, disruptinggenes such as NLRC5, TAP1, and B2M in the grafts can cause lack offunctional expression of MHC class Ion graft cells including on isletbeta cells, thereby interfering with the posttransplant activation ofautoreactive CD8+ T cells. Thus, this can protect the transplant, e.g.,transplanted islet beta cells, from the cytolytic effector functions ofautoreactive CD8+ T cells.

Inducing the Tolerance of Transplant Grafts in a Recipient UsingTolerizing Vaccines

A tolerizing vaccine comprising ECDI-treated cells can be administeredbefore, after, and/or during transplant of donor cells, organs, and/ortissues to induce donor-specific tolerance in a recipient. As an exampleshow in FIG. 4, a pig islet is transplanted to a recipient (e.g., ahuman or a non-human animal) on day 0. Apoptotic cells (e.g., tolerizingvaccine) derived from the same donor pig can be first administered 7days before islet transplant (day −7) for inducing tolerance to thexenograft (e.g., pig islet from the same donor). An additionaltolerizing vaccine can be administered 1 day after islet transplant(day 1) to booster tolerance (FIG. 4). Furthermore, administration of atolerizing vaccine can be accompanied by administration of transientimmunosuppression (FIG. 4). In some cases, a tolerizing vaccinecomprising ECDI-treated cells can be administered on or on about day−100, day −90, day −80, day −70, day −60, day −50, day −40, day −30, day−20, day −15, day −14, day −13, day −12, day −11, day −10, day −9, day−8, day −7, day −6, day −5, day −4, day −3, day −2 or day −1, relativeto transplant of donor cells, organs, and/or tissues on day 0, e.g., onor on about day −100 to −50; −50 to −40; −40 to −30; −30 to −20; −20 to−10; −10 to −5; −7 to −1. For example, a tolerizing vaccine comprisingECDI-treated cells can be administered 7 days before (e.g., day −7)transplant of donor cells, organs, and/or tissues. In some cases, atolerizing vaccine comprising ECDI-treated cells can be administered onthe same day (e.g., day 0) as transplant of donor cells, organs, and/ortissues. In some cases, ECDI-treated cells can be administered on or onabout day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8,day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative totransplant of donor cells, organs, and/or tissues on day 0. For example,a tolerizing vaccine comprising ECDI-treated cells can be administeredon 1 day after (e.g., day 1) transplant of donor cells, organs, and/ortissues. In some cases, the tolerizing vaccine can be administeredbefore and after the transplantation of donor cells, organs, and/ortissues.

Genetically modified cells, tolerizing vaccines and antibodies can beused together to suppress transplant rejection. FIG. 5 demonstrates anexemplary approach to preventing rejection and/or extending survival ofa graft (e.g., a xenograft). The approach can integrate: i) geneticengineering of the graft donor; ii) genetic engineering of the vaccinedonor, and iii) the administration of the genetically engineeredtolerizing vaccine (apoptotic cells alone or with non-apoptotic cells),and the graft under the cover of the transient immunosuppression. Agraft donor and a vaccine donor can have the same genotype.Alternatively, a graft donor and a vaccine donor can have differentgenotypes. In some cases, a graft donor can comprise reduced expressionof NLRC5, C3, CXCL10, and GGTA1, and transgenes comprisingpolynucleotides encoding HLA-G (e.g., HLA-G1) or HLA-E. In some cases, agraft donor can comprise reduced expression of TAP1, C3, CXCL10, andGGTA1, and transgenes comprising polynucleotides encoding HLA-G (e.g.,HLA-G1) or HLA-E. In some cases, a graft donor can comprise reducedexpression of NLRC5 and TAP1, C3, CXCL10, and GGTA1, and transgenescomprising polynucleotides encoding HLA-G (e.g., HLA-G1) or HLA-E. Avaccine donor can have reduced expression of GGTA1, CMAH, B4GALNT2and/or transgenes comprising polynucleotides encoding HLA-G (e.g.,HLA-G1), CD47 (e.g., human CD47), PD-L1 (e.g., human PD-L1), and PD-L2(e.g., human PD-L2). A vaccine donor can have reduced expression ofGGTA1, CMAH, B4GALNT2 and/or transgenes comprising polynucleotidesencoding HLA-E, CD47 (e.g., human CD47), PD-L1 (e.g., human PD-L1), andPD-L2 (e.g., human PD-L2). The vaccines in some instances can be givento a transplant recipient before (e.g., on day −7) and/or after (e.g.,on day 1). Other immunosuppression reagents, e.g., one or more ofanti-CD40 antibodies, anti-CD20 antibodies, rapamycin, compstatin,anti-IL-6R antibodies, and sTNFR, a nitrogen mustard alkylating agent(e.g., cyclophosphamide) can also be given to the subject before and/orafter transplant.

In addition to the genetically modified cells, tissues, organs,tolerizing vaccines and anti-CD40 antibodies disclosed herein, one ormore additional immunosuppression agents can also be administered to asubject receiving the genetically modified cells, tissues, organs,tolerizing vaccines and/or anti-CD40 antibodies. The additionalimmunosuppression agent can be administered to a subject, e.g., toenhance the tolerogenic efficacy of a tolerizing vaccine in the subject.The additional immunosuppression agent can include a B-cell depletingantibody, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, anitrogen mustard alkylating agent (e.g., cyclophosphamide), a complementC3 or C5 inhibitor, or any combination thereof.

The additional immunosuppression agent, e.g., can be a nitrogen mustardalkylating agent. For example, the additional immunosuppression agentcan be cyclophosphamide.

The additional immunosuppression agent can be administered before,after, and/or during the administration of a tolerizing vaccine. In somecases, the additional immunosuppression agent can be administeredbetween day −100 and day 0, e.g., on day −90, day −80, day −70, day −60,day −50, day −40, day −30, day −20, day −15, day −14, day −13, day −12,day −11, day −10, day −9, day −8, day −7, day −6, day −5, day −4, day−3, day −2 or day −1, relative to the administration of a tolerizingvaccine. In some cases, the additional immunosuppression agent can beadministered on or on about day −100 to −50; −50 to −40; −40 to −30; −30to −20; −20 to −10; −10 to −5; −7 to −1, relative to the administrationof a tolerizing vaccine. In some cases, the additional immunosuppressionagent can be administered between day 0 and day 100, e.g., on day 100,day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15,day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day5, day 4, day 3, day 2 or day 1 relative to the administration of atolerizing vaccine. For example, the immunosuppression agent can beadministered on or on about day 100 to 50; 50 to 40; 40 to 30; 30 to 20;20 to 10; 10 to 5; 7 to 1, relative to the administration of atolerizing vaccine. In some cases, the additional immunosuppressionagent can be administered on the day when a tolerizing vaccine isadministered. In other cases, the additional immunosuppression can beadministered before and after the administration of the tolerizingvaccine. For example, cyclophosphamide can be administered on or onabout day 3 after the administration of a tolerizing vaccine.

A tolerogenic efficacy regulator (e.g., cyclophosphamide) can beadministered at dose from or from about 5 to 100 mg/kg/day. The unit“mg/kg/day” can refer to the number of milligrams of the tolerogenicefficacy regulator given per kilogram of the subject's body weight perday. In some cases, a tolerogenic efficacy regulator (e.g.,cyclophosphamide) can be administered at a dose of from or from about 20mg/kg/day to 100 mg/kg/day; 30 mg/kg/day to 90 mg/kg/day; 40 mg/kg/dayto 80 mg/kg/day; 50 mg/kg/day to 70 mg/kg/day; 50 mg/kg/day to 60mg/kg/day; or 40 mg/kg/day to 60 mg/kg/day.

Cells (e.g., splenocytes) can be treated with ECDI in the presence ofsuitable antigen(s) and/or epitope(s) (e.g., CD4). ECDI-treatment canresult in coupling of antigen(s) and/or epitope(s) to ECDI-treatedcells. Other conjugates such as hexamethylene diisocyanate,propyleneglycol di-glycidylether which contain 2 epoxy residues, andepichlorohydrin can also be used to treat cells and couple antigens(s)and/or epitope(s) to make cells for tolerizing vaccines.

Antigen-coupled and/or epitope-coupled cells (e.g., ECDI-inducedcoupling) can be administered before, during, and/or afteradministration of donor transplant cells, organs, and/or tissues toinduce tolerance for the cells, organs, and/or tissues in a recipient(e.g., a human or a non-human animal). In some cases, antigen-coupledand/or epitope-coupled cells can be administered on day −100, day −90,day −80, day −70, day −60, day −50, day −40, day −30, day −20, day −15,day −14, day −13, day −12, day −11, day −10, day −9, day −8, day −7, day−6, day −5, day −4, day −3, day −2 or day −1, relative to transplant ofdonor cells, organs, and/or tissues on day 0. In some cases, theantigen-coupled and/or epitope-coupled cells can be administered on orabout on day −100 to −50; −50 to −40; −40 to −30; −30 to −20; −20 to−10; −10 to −5; −7 to −1, relative to transplant of donor cells, organs,and/or tissues on day 0. For example, antigen-coupled and/orepitope-coupled cells can be administered 7 days before (e.g., day −7)transplant of donor cells, organs, and/or tissues. In some cases,antigen-coupled and/or epitope-coupled cells can be administered on thesame day (e.g., day 0) as the transplant of donor cells, organs, and/ortissues. In some cases, antigen-coupled and/or epitope-coupled cells canbe administered on day 100, day 90, day 80, day 70, day 60, day 50, day40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative totransplant of donor cells, organs, and/or tissues on day 0. For example,the antigen-coupled and/or epitope-coupled cells can be administered onor on about day 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to5; 7 to 1, relative to transplant of donor cells, organs, and/or tissueson day 0. For example, antigen-coupled and/or epitope-coupled cells canbe administered on 1 day after (e.g., day 1) transplant of donor cells,organs, and/or tissues.

ECDI-treated cells, antigen-coupled cells, and/or epitope-coupled cellscan be administered to a recipient prior to transplantation of donorcells, organs, and/or tissues to a recipient. ECDI-treated cells,antigen-coupled cells, and/or epitope-coupled cells can beco-administered to a recipient prior to transplantation of donor cells,organs, and/or tissues to a recipient. ECDI-treated cells,antigen-coupled cells, and/or epitope-coupled cells can be administeredto a recipient following transplantation of donor cells, organs, and/ortissues to a recipient. Administration of ECDI-treated cells,antigen-coupled cells, and/or epitope-coupled cells to a transplantrecipient before, during, and/or after transplantation can result inincreased tolerance of transplanted cells, organs, and/or tissues. Forexample, ECDI-treated cells, antigen-coupled cells, and/orepitope-coupled cells can increase initial tolerance, long-termtolerance, and/or total acceptance of transplanted cells, organs, and/ortissues. In some cases, administering ECDI-treated cells (e.g.epitope-coupled cells) to a transplant recipient can result in toleranceof transplanted materials without additional immunosuppression oranti-rejection therapies.

Tolerizing vaccines can reduce the dose or duration of immunosuppressionrequired to prevent rejection of cells, organs, and/or tissues.Tolerizing vaccines can reduce the dose of immunosuppression required byat least or at least about 5%. For example, Tolerizing vaccines reducethe dose of immunosuppression required by at least or at least about 5%,10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%, e.g., by at least or at least about 5 to 25; 25 to 50; 50 to 75;75 to 85; 85 to 90; 90 to 95; 95 to 100%. In some cases, a transplantrecipient can require no immunosuppression after administration of atolerizing vaccine. The term “reduce” and its grammatical equivalents asused herein can refer to using less immunosuppression compared to arequired dose of immunosuppression when one or more wild-type cells,organs, and/or tissues is transplanted into a recipient (e.g., a humanor a non-human animal). The term “reduce” can also refer to using lessimmunosuppressive drug(s) or agent(s) compared to a required dose ofimmunosuppression when one or more wild-type cells, organs, and/ortissues is transplanted into a recipient (e.g., a human or a non-humananimal).

A recipient (e.g., a human or a non-human animal) can require a reduceddose of immunosuppression for at least or at least about 1, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, or 100 days after transplantation, e.g., forat least or at least about 1 to 5; 5 to 10; 10 to 20; 20 to 30; 30 to60; 60 to 100 days. A recipient (e.g., a human or a non-human animal)can require a reduced dose of immunosuppression for at least or at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months aftertransplantation, e.g., for at least or at least about 1 to 2; 2 to 3; 3to 6; 6 to 9; 9 to 12 months after transplantation. A recipient (e.g., ahuman or a non-human animal) can require a reduced dose ofimmunosuppression for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, or 30 years after transplantation, e.g. for at leastor at least about 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to15; 15 to 20; 20 to 25; 25 to 30 years after transplantation. In somecases, a recipient (e.g., a human or a non-human animal) can require areduced dose of immunosuppression for up to the lifetime of therecipient.

A recipient (e.g., a human or a non-human animal) can require noimmunosuppression for at least or at least about 1, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, or 100 days after transplantation, e.g., for atleast or at least about 1 to 5; 5 to 10; 10 to 20; 20 to 30; 30 to 60;60 to 100 days. A recipient (e.g., a human or a non-human animal) canrequire a reduced dose of immunosuppression for at least or at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months aftertransplantation e.g., for at least or at least about 1 to 2; 2 to 3; 3to 6; 6 to 9; 9 to 12 months after transplantation. A recipient (e.g., ahuman or a non-human animal) can require a reduced dose ofimmunosuppression for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, or 30 years after transplantation, e.g. for at leastor at least about 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to15; 15 to 20; 20 to 25; 25 to 30 years after transplantation. In somecases, a recipient (e.g., a human or a non-human animal) can require noimmunosuppression for up to the lifetime of the recipient.

Immunosuppression described herein can refer to the immunosuppressionadministered immediately before, after, and/or during transplantation.Immunosuppression described herein can also refer to the maintenanceimmunosuppression administered at least or at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days (e.g., forat least or at least about 1 to 5; 5 to 10; 10 to 20; 20 to 30; 30 to60; 60 to 100 days) or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30years (e.g., for at least or at least about 1 to 2; 2 to 3; 3 to 4; 4 to5; 1 to 5; 5 to 10; 10 to 15; 15 to 20; 20 to 25; 25 to 30 years) aftertransplantation. Tolerizing vaccines can increase survival of cells,organs, and/or tissues without need for maintenance immunosuppression.

Immunosuppression can be used in immunosuppressive therapy to suppresstransplant rejection in a recipient. Immunosuppressive therapy cancomprise any treatment that suppresses transplant rejection in arecipient (e.g., a human or a non-human animal). Immunosuppressivetherapy can comprise administering immuno-suppressive drugs.Immunosuppressive drugs that can be used before, during, and/or aftertransplant include, but are not limited to, MMF (mycophenolate mofetil(Cellcept)), ATG (anti-thymocyte globulin), anti-CD154 (CD4OL),alemtuzumab (Campath), anti-CD20 (rituximab), anti-IL-6R antibody(tocilizumab, Actemra), anti-IL-6 antibody (sarilumab, olokizumab),CTLA4-Ig (Abatacept/Orencia), belatacept (LEA29Y), sirolimus (Rapimune),tacrolimus (Prograf), daclizumab (Ze-napax), basiliximab (Simulect),infliximab (Remicade), cyclosporin, deoxyspergualin, soluble complementreceptor 1, cobra venom factor, compstatin, anti C5 antibody(eculizumab/Soliris), methylprednisolone, FTY720, everolimus,anti-CD154-Ab, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122antibody, and human anti-CD154 monoclonal antibody. One or more than oneimmunosuppressive agents/drugs can be used together or sequentially. Oneor more than one immunosuppressive agents/drugs can be used forinduction therapy or for maintenance therapy. The same or differentdrugs can be used during induction and maintenance stages. For example,daclizumab (Zenapax) is used for induction therapy and tacrolimus(Prograf) and sirolimus (Rapimune) is used for maintenance therapy. Inanother example, daclizumab (Zenapax) is used for induction therapy andlow dose tacrolimus (Prograf) and low dose sirolimus (Rapimune) is usedfor maintenance therapy. Immunosuppression can also be achieved usingnon-drug regimens including, but not limited to, whole body irradiation,thymic irradiation, and full and/or partial splenectomy. Thesetechniques can also be used in combination with one or moreimmuno-suppressive drug.

Antibody Treatment

Both allografts and xenografts that escape fulminant, hyperacute, and/oracute vascular rejection are subjected to T cell mediated rejection.CD4⁺ and CD8⁺ T lymphocytes contribute to rejection. These T cells canbe activated via the direct pathway of immune recognition involvingpresentation by donor antigen presenting cells to T cells or via theindirect pathway involving presentation of internalized soluble donorantigen by host antigen presenting cells. CD8⁺ T cells are mainmediators of rejection. B cells promote proliferation of activatedanti-donor CD4⁺ T cells, survival of anti-donor CD8⁺ T cells, and T cellmemory generation by mechanisms such as antigen presentation, cytokineproduction, and co-stimulation. The compositions and methods disclosedherein can be used to reduce a recipient's direct immune responses,indirect immune responses, or both to a cell, tissue or organtransplanted from a donor. The methods of treatment as described hereincan comprise providing ECDI-treated cells (e.g., tolerizing vaccine) andone or more biological or chemical substances to a human. For example,ECDI-treated cells can be porcine cells, e.g., porcine splenocytes.

One or more biological or chemical substances can be an antibody. Anantibody can be an anti-CD40 or anti-IL-6R. An anti-CD40 antibody can bean anti-CD40 Ab 2C10 antibody, an anti-CD40 mAb ASKP1240 (4D11) (e.g.,as described in Watanabe et al., “ASKP1240, a fully human anti-CD40monoclonal antibody, prolongs pancreatic islet allograft survival innonhuman primates,” Am J Transplant. 13(8):1976-88 (2013), or ananti-CD40 mAb CFZ533 (as described in Corodoba et al., “A Novel,Blocking, Fc-Silent Anti-CD40 Monoclonal Antibody Prolongs NonhumanPrimate Renal AllograftSurvival in the Absence of B Cell Depletion,” AmJ Transplant, 15(11):2825-36 (2015).

Methods described herein for immunotolerizing a recipient (e.g., a humanor a non-human animal) for transplantation (e.g., xenotransplantation)can comprise providing to a recipient (e.g., a human or a non-humananimal) two or more biological or chemical substances selected from agroup consisting of: ECDI-treated cells, B cell depleting antibodies,antagonistic anti-CD40 antibodies, mTOR inhibitors, and TNF-alphainhibitors, and IL-6 inhibitors, or any combination thereof. Methodsherein for prolonging transplantation survival in a recipient (e.g., ahuman or a non-human animal) can comprise administering to the recipient(e.g., a human or a non-human animal) two or more biological substancesselected from the group consisting of ECDI-treated cells, anti-CD40 Ab2C10 antibody, sTNFR, anti-IL-6R antibody, or any combination thereof.For example, the methods can comprise providing to a recipient (e.g., ahuman or a non-human animal) ECDI-treated cells, where the ECDI-treatedcells are disclosed herein. The methods can comprise providing to arecipient (e.g., a human or a non-human animal) B cell depletingantibodies, for example, rituximab. The methods can comprise providingto a recipient (e.g., a human or a non-human animal) antagonisticanti-CD40 antibodies, for example, humanized 2C10. The methods cancomprise providing to a recipient (e.g., a human or a non-human animal)mTOR inhibitors, for example, rapamycin. The methods can compriseproviding to a recipient (e.g., a human or a non-human animal) TNF-alphainhibitors, for example, sTNFR. sTNFR can also be tocilizumab oretanercept. The methods can comprise providing to a recipient (e.g., ahuman or a non-human animal) an IL-6 inhibitor, for example, ananti-IL-6R antibody. In some cases, the methods can comprise providingto a recipient (e.g., a human or a non-human animal) an antibody (e.g.,a monoclonal antibody) targeting a non-redundant epitope on antigenpresenting cells (APC). In some cases, the methods can compriseadministering pharmaceutical agents that inhibit T cell activation, Bcell activation, dendritic cell activation, or any combination thereof.

The present disclosure can also provide a kit comprising two or more ofthe following: a splenocyte; anti-CD40 Ab 2C10 antibody; sTNFR; andanti-IL-6R antibody. For example, a kit can comprise a splenocyte andanti-CD40 Ab 2C10 antibody. A kit can comprise a splenocyte and sTNFR. Akit can comprise a splenocyte and anti-IL-6R antibody. A kit cancomprise an anti-CD40 Ab 2C10 antibody and sTNFR. A kit can comprise ananti-CD40 Ab 2C10 antibody and anti-IL-6R antibody. A kit can comprise asTNFR and anti-IL-6R antibody. A kit can comprise a splenocyte,anti-CD40 Ab 2C10 antibody and sTNFR. A kit can comprise a splenocyte,anti-CD40 Ab 2C10 antibody and anti-IL-6R antibody. A kit can comprise asplenocyte, sTNFR and anti-IL-6R antibody. A kit can comprise anti-CD40Ab 2C10 antibody, sTNFR and anti-IL-6R antibody. A kit can comprise asplenocyte; anti-CD40 Ab 2C10 antibody; sTNFR; and anti-IL-6R antibody.A kit can further comprise a reagent for ECDI fixation.

The methods herein can comprise ECDI-treated cells, such as ECDI-treatedsplenocytes. In some cases, the methods can comprise providing to arecipient (e.g., a human or a non-human animal) ECDI-treated splenocytesand anti-CD40 Ab 2C10 antibody. In some cases, the methods can compriseproviding to a recipient (e.g., a human or a non-human animal)ECDI-treated splenocytes and sTNFR. In some cases, the methods cancomprise providing to a recipient (e.g., a human or a non-human animal)ECDI-treated splenocytes and anti-IL-6R antibody. In some cases, themethods can comprise providing to a recipient (e.g., a human or anon-human animal) anti-CD40 Ab 2C10 antibody and sTNFR. In some cases,the methods can comprise providing to a recipient (e.g., a human or anon-human animal) anti-CD40 Ab 2C10 antibody and anti-IL-6R antibody. Insome cases, the methods can comprise providing to a recipient (e.g., ahuman or a non-human animal) ECDI-treated splenocytes, anti-CD40 Ab 2C10antibody, and sTNFR. In some cases, the methods can comprise providingto a recipient (e.g., a human or a non-human animal) ECDI-treatedsplenocytes, anti-CD40 Ab 2C10 antibody, and anti-IL-6R. In some cases,the methods can comprise providing to a recipient (e.g., a human or anon-human animal) ECDI-treated splenocytes, sTNFR, and anti-IL-6Rantibody. In some cases, the methods can comprise providing to arecipient (e.g., a human or a non-human animal) ECDI-treatedsplenocytes, anti-CD40 Ab 2C10 antibody, sTNFR, and anti-IL-6R antibody.In some cases, the methods can comprise providing to a recipient (e.g.,a human or a non-human animal) ECDI-treated splenocytes, and an antibody(e.g., a monoclonal antibody) targeting a non-redundant epitope onantigen presenting cells (APC).

A donor (e.g., a donor for a transplant graft and/or a cell in atolerizing vaccine) can be a mammal. A donor of allografts can be anunmodified human cell, tissue, and/or organ, including but not limitedto pluripotent stem cells. A donor of xenografts can be any cell,tissue, and/or organ from a non-human animal, such as a mammal. In somecases, the mammal can be a pig.

The methods herein can further comprise diagnosing a recipient (e.g., ahuman or a non-human animal) with a disease. For example, a disease isdiabetes, including but not limited to, type 1, type 2, gestational,surgical, cystic fibrosis-related diabetes, or mitochondrial diabetes.In some cases, a disease can be hereditary diabetes or a type ofhereditary diabetes.

The methods herein can comprise administering ECDI-treated cells before,after, and/or during transplant of donor cells, organs, and/or tissuesto induce donor-specific tolerance in a recipient. In some cases,ECDI-treated cells can be administered on or on about day −100, day −90,day −80, day −70, day −60, day −50, day −40, day −30, day −20, day −15,day −14, day −13, day −12, day −11, day −10, day −9, day −8, day −7, day−6, day −5, day −4, day −3, day −2 or day −1, relative to transplant ofdonor cells, organs, and/or tissues on day 0. In some cases, theantigen-coupled and/or epitope-coupled cells can be administered on orabout on day −100 to −50; −50 to −40; −40 to −30; −30 to −20; −20 to−10; −10 to −5; −7 to −1, relative to transplant of donor cells, organs,and/or tissues on day 0. For example, ECDI-treated cells can beadministered 7 days before (e.g., day −7) transplant of donor cells,organs, and/or tissues. In some cases, ECDI-treated cells can beadministered on the same day (e.g., day 0) as transplant of donor cells,organs, and/or tissues. In some cases, ECDI-treated cells can beadministered on or on about day 100, day 90, day 80, day 70, day 60, day50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1,relative to transplant of donor cells, organs, and/or tissues on day 0.For example, the antigen-coupled and/or epitope-coupled cells can beadministered on or on about day 100 to 50; 50 to 40; 40 to 30; 30 to 20;20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells,organs, and/or tissues on day 0. For example, ECDI-treated cells can beadministered on 1 day after (e.g., day 1) transplant of donor cells,organs, and/or tissues.

The methods herein can comprise administering at least or at least about0.25×10⁹ ECDI-treated cells (e.g., donor splenocytes) per kg recipientbody weight. For example, at least or at least about 1×10⁷, 1×10⁸,0.25×10⁹, 0.50×10⁹, 0.75×10⁹, 1.00×10⁹, 1.25×10⁹, 1.50×10⁹, 1.75×10⁹ or2×10⁹ ECDI-treated cells (e.g., donor splenocytes) per kg recipient bodyweight ECDI-treated cells can be administered. ECDI-treated cells canalso be splenic B cells. The methods herein can comprise administeringfrom or from about 1×10⁸ to 2×10⁹, e.g., 1×10⁸ to 2×10⁸, 1×10⁸ to 3×10⁸,1×10⁸ to 4×10⁸, 1×10⁸ to 5×10⁸, 1×10⁸ to 1×10⁹, ECDI-treated cells(e.g., donor splenocytes) per kg recipient body weight.

Donor splenocytes can be freshly isolated. Alternatively, ECDI-treatedcells can be ex-vivo expanded. In some cases, donor splenocytes compriseat least or at least about 10%, e.g., 25%, CD21 positive SLA Class IIpositive B cells. For example, donor splenocytes comprise at least or atleast about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% CD21positive SLA Class II positive B cells, e.g., at least or at least about10 to 20; 20 to 30; 30 to 40; or 40 to 50%. In some cases, splenic Bcells comprise at least or at least about 60%, e.g., 90%, CD21 positiveSLA Class II positive B cells. For example, splenic B cells comprise atleast or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD21positive SLA Class II positive B cells e.g., at least or at least about60 to 70; 70 to 80; 80 to 90; or 90 to 95%. In some cases, donorsplenocytes comprise from or from about 50% to 100%, e.g., from or fromabout 60% to 100% or 80% to 100%, CD21 positive SLA Class II positive Bcells.

ECDI-treated cells can be given intravenously. ECDI-treated cells areinfused intravenously. In some cases, ECDI-treated cells can be givenintravenously in a volume of at least or at least about 1 ml, 2 ml, 3ml, 4 ml, 5 ml, 10 ml, 20 ml, 30 ml, 40 ml or 50 ml per kg recipientbody weight, e.g., at least or at least about 1 to 2; 2 to 3; 3 to 4; 4to 5; 1 to 5; 5 to 10; 10 to 20; 20 to 30; 30 to 40; or 40 to 50. Forexample, ECDI-treated cells are given intravenously in a volume of 7 mlper kg recipient body weight.

The methods herein can further comprise treating a disease bytransplanting one or more donor cells to an immunotolerized recipient(e.g., a human or a non-human animal).

The methods can comprise providing cells (e.g., ECDI-treated cells) oneor more disrupted genes selected from NOD-like receptor family CARDdomain containing 5 (NLRC5), Transporter associated with antigenprocessing 1 (TAP1), GGTA1, B4GALNT2, CMAH, C-X-C motif chemokine 10(CXCL10), MHC class I polypeptide-related sequence A (MICA), MHC class Ipolypeptide-related sequence B (MICB), or class II majorhistocompatibility complex transactivator (CIITA). ECDI-treated cellscan be derived from the same donor. Furthermore, ECDI-treated cells canfurther comprise one or more transgenes selected from ICP47, CD46, CD55,CD59, or any combination thereof. In some cases, donor cells can beislet cells. In some cases, the one or more disrupted gene does notinclude GGTA1.

Antagonistic anti-CD40 monoclonal antibody 2C10 can be given incombination with other immunotherapy (sTNFR, anti-IL-6R, mTOR inhibitor,with and without anti-CD20 monoclonal antibodies, and with or withoutCTLA4-Ig) and with or without intravenous infusion of donor apoptoticcells. This treatment can facilitate remarkable and unprecedented isletallograft and pig islet xenograft survival in primates, e.g., monkeys.For example, most remarkable is the maintenance of excellent bloodglucose control in transplanted monkeys despite discontinuation ofexogenous insulin and all immunosuppression on or on about day 21posttransplant. Examples include the maintenance of excellent isletallograft function in 3 of 4 monkeys for at least or at least about 200days (2 without and 1 with administration of donor apoptotic cells) andthe maintenance of excellent islet xenograft function in 1 of 1 monkeysfor at least or at least about 100 days (with administration of donorapoptotic cells).

Other methods of use can include i) transient or infrequent use ofanti-CD40 monoclonal antibody 2C10 or similar antibodies for preventionof rejection of genetically modified grafts, ii) transient or infrequentuse of anti-CD40 monoclonal antibody 2C10 or similar antibodies intransplantation in conjunction with other immunotherapy targetinginflammation (e.g., complement inhibitors and cytokine and chemokineinhibitors such as the IL-8 inhibitor reparaxin), and the use ofanti-CD40 monoclonal antibody 2C10 or similar antibodies for preventionof stem cell-derived cellular grafts such as functional human islet betacells.

The methods herein can comprise administering one or more dose ofanti-CD40 antibody to a recipient before, after, and/or duringtransplant of donor cells, organs, and/or tissues to inducedonor-specific tolerance in a recipient. In some cases, a first dose ofanti-CD40 antibody can be given on or on about day −100, day −90, day−80, day −70, day −60, day −50, day −40, day −30, day −20, day −15, day−14, day −13, day −12, day −11, day −10, day −9, day −8, day −7, day −6,day −5, day −4, day −3, day −2 or day −1, relative to transplant ofdonor cells, organs, and/or tissues on day 0. In some cases, a firstdose of anti-CD40 antibody can be given on or on about day −100 to −50;−50 to −40; −40 to −30; −30 to −20; −20 to −10; −10 to −5; −7 to −1,relative to transplant of donor cells, organs, and/or tissues on day 0.For example, a first dose of anti-CD40 antibody can be given 8 days(e.g., day −8) before transplant of donor cells, organs, and/or tissues.

Different doses of anti-CD40 antibody can be given to a recipientbefore, after, and/or during transplant of donor cells, organs, and/ortissues to induce donor-specific tolerance in a recipient. In somecases, a first dose of anti-CD40 antibody can comprise at least or atleast about 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg,80 mg, 90 mg, 100 mg, or 200 mg of the anti-CD40 antibody per kgrecipient body weight. In certain cases, a first dose of anti-CD40antibody can comprise at least or at least about 30 mg, 40 mg, 50 mg, 60mg, 70 mg of the anti-CD40 antibody per kg recipient body weight. Insome cases, a first dose of anti-CD40 antibody can comprise from or fromabout 1 mg to 200 mg, e.g., from or from about 20 mg to 100 mg; 30 mg to80 mg; 30 mg to 70 mg; 40 mg to 70 mg; 40 mg to 60 mg; 50 mg to 70 mg;or 60 mg to 80 mg of the anti-CD40 antibody per kg recipient bodyweight.

FIG. 6 demonstrates an exemplary protocol for transplant rejectionprophylaxis in pig-to-cynomolgus monkey islet xenotransplantation. For acynomolgus monkey transplanted with 25,000 islet equivalents/kg on day 0(the day of transplantation), the protocol for transplant rejectionprophylaxis can include: administering ECDI-fixed, apoptotic donorsplenocytes on days −7 and 1, administering an α-CD40 (e.g., 2C10) 50mg/kg on days −8, −1, 7, 14, administering an α-CD20 antibody (e.g.,rituximab) 20 mg/kg on days −10, −3, 5, and 12, administering rapamycin(target trough 15-25 ng/mL), administering sTNFR (1 mg/kg on days −7 and0, 0.5 mg/kg on days 3, 7, 10, 14, and 21), and administering an α-IL-6Rantibody on days −7, 0, 7, 14, and 21 to the cynomolgus monkey.

EXAMPLES Example 1: Generating Plasmids Expressing Guide RNA forDisrupting GGTA1, CMAH, NLRC5, B4GALNT2, and/or C3 Genes in Pigs

Genetically modified pigs will provide transplant grafts that induce lowor no immuno-rejection in a recipient, and/or cells as tolerizingvaccines that enhance immuno-tolerization in the recipient. Such pigswill have reduced expression of any genes that regulate MHC molecules(e.g., MHC I molecules and/or MHC II molecules) compared to anon-genetically modified counterpart animal. Reducing expression of suchgenes will result in reduced expression and/or function of MHCmolecules. These genes will be one or more of the following: componentsof an MHC I-specific enhanceosome, transporters of a MHC I-bindingpeptide, natural killer group 2D ligands, CXCR 3 ligands, C3, and CIITA.Additionally or alternatively, such pigs will comprise reduced proteinexpression of an endogenous gene that is not expressed in human (e.g.,CMAH, GGTA1 and/or B4GALNT2). For example, the pigs will comprisereduced protein expression of one or more of the following: NLRC5, TAP1,C3, CXCL10, MICA, MICB, CIITA, CMAH, GGTA1 and/or B4GALNT2. In somecases, pigs will comprise reduced protein expression of NLRC5, C3,CXCL10, CMAH, GGTA1 and/or B4GALNT2.

This example shows exemplary methods for generating plasmids fordisrupting GGTA1, CMAH, NLRC5, B4GALNT2, and/or C3 genes in pigs usingthe CRISPR/cas9 system. The plasmids were generated using the px330vector, which simultaneously expressed a Cas9 DNA endonuclease and aguide RNA.

The px330-U6-Chimeric_BB-CBh-hSpCas9 (#42230) plasmid was obtained fromAddgene in a bacterial stab culture format. The stab culture wasstreaked onto a pre-warmed LB agar with ampicillin plate and incubatedat 37° C. overnight. The next day, a single colony was selected andinoculated in a liquid LB overnight culture with ampicillin (5 mL formini-prep, or 80-100 mL for maxi-prep). Mini-prep was performed usingQiagen kits according to manufacturer's instructions. Plasmid was elutedin nuclease free water and stocks were stored at −20° C. Theoligonucleotides designed for targeting GGTA1, CMAH, NLRC5, C3, andB4GALNT2 are shown in Table 6. The oligonucleotides were synthesized byIDT. FIGS. 7A-7E, 8A-8E, 9A-9E, 10A-10E, and 11A-11E, show the cloningstrategies for cloning plasmids targeting GGTA1 (i.e., px330/Ga12-1)(FIGS. 7A-7E), CMAH (i.e., px330/CM1F) (FIGS. 8A-8E), NLRC5 (i.e.,px330/NL1_First) (FIGS. 9A-9E), C3 (i.e., px330/C3-5) (FIGS. 10A-10E),and B4GALNT2 (i.e., px330/B41_second) (FIGS. 11A-11E). The constructedpx330 plasmids were validated by sequencing using sequencing primersshown in Table 7. Oligonucleotides were re-suspended at 100 μM withnuclease free water and stored in the −20° C. freezer.

Vector digestion: The px330 vectors were digested in a reaction solutioncontaining 5 μg px330 stock, 5 μL 10× FastDigest Reaction Buffer, 35 μLnuclease free water, and 5 μL FastDigest BbsI enzyme (Cutsite: GAAGAC).The reaction solution was incubated at 37° C. for 15 minutes, the heatinactivated at 65° C. for 15 minutes. To desphosphorylate the vector,0.2 μL (2 U; 1 U/1 pmol DNA ends) CIP was added and the resultingmixture was incubated at 37° C. for 60 minutes. The linearized plasmidwas purified using Qiagen PCR Cleanup kit, and eluted with nuclease freewater and stored at −20° C. until use.

Oligonucleotides Annealing and phosphorylation: a solution was made bymixing 1 μL 100 uM Forward oligonucleotide, 1 μL 100 uM Reverseoligonucleotide, 1 μL 10×T4 Ligase Buffer, 6 μL nuclease free water, 1μL Polynucleotide Kinase (PNK). The resulting solution was incubated ona thermal cycler running the following program: 37° C. for 30 min, 95°C. for 5 min, ramp down to 25° C. at 0.1° C./second.

Ligation Reaction: a solution was made by mixing diluted annealedoligonucleotides 1:250 with nuclease free water, 2 μL diluted annealedoligonucleotides, 100 ng linearized/dephosphorylated px330 vector, 5 μL10×T4 Ligase Buffer, nuclease free water to bring to 50 μL final volume,and 2.5 μL T4 DNA Ligase. The solution was incubated at room temp for 4hours, then heat inactivated at 65° C. for 10 minutes.

Transformation: TOP10 E. coli vials were thawed from −80° C. freezer onice for 15 minutes prior to transformation. 2 μL of the ligationreaction product was added to the cells and mixed by gently flicking thetubes. The tubes were incubated on ice for 5 minutes, heat shocked in42° C. water bath for 30 seconds, and placed back on ice for additional2 minutes after heat shock. 50 μL of transformed cells were plated ontoan LB agar with ampicillin plate and spread with pipette tip. The plateswere incubated at 37° C. overnight.

Colony PCR screening for correctly inserted oligonucleotides: 3×colonies were selected from the plate and labeled 1-3 on bottom ofplate. Master mix for PCR reaction was prepared by mixing 15 μL 10×Standard Taq Reaction Buffer, 3 μL 10 mM dNTP mix, 0.5 μL 100 uMpx330-F1 primer (SEQ ID No. 125 in Table 7), 0.5 μL 100 uM px330-R1primer (SEQ ID No. 126 in Table 7), 130 μL nuclease free water, and 1 μLStandard Taq Polymerase. Master mix was vortexed briefly, thenaliquotted 50 μL to 3×PCR tubes labeled 1-3. A pipette tip was dabbedinto colony #1 on the agar plate and then pipetted up and down in PCRtube #1. Repeated for each colony being screened using a fresh tip foreach colony. Tubes were placed in thermal cycler to run the followingprogram: 95° C. for 5 min, 95° C. for 30 seconds, 52° C. for 30 seconds,68° C. for 30 seconds, cycle step 2-4 for 30 cycles, 68° C. for 5 min,hold at 4° C. until use. PCR Cleanup was performed using Qiagen PCRCleanup Kit and followed manufacturer's protocol. The product was elutedin nuclease free water.

Preparing samples for sequencing: a solution was made by mixing 120 ngPCR product, 6.4 pmols px330-F1 primer (1 μL of 6.4 μM stock), andnuclease free water that brought the final volume to 12 After thesequence data was obtained, correct sequence inserts were identified.Glycerol stocks of colonies with correct inserts were prepared. On theLB agar plate labeled during colony PCR with #1-3, the correctlyinserted colonies were inoculated in 5 mL LB medium with ampicillin bydabbing with a pipette tip and ejecting into the tube of medium. Liquidculture was grown out until an OD was reached between 1.0 and 1.4. 500μL of bacterial culture was added to 500 μL of sterile 50% glycerol in acryovial and placed immediately on dry ice until transfer to −80° C.freezer.

TABLE 6Exemplary oligonucleotides for making guide RNA constructs targeting GGTA1,CMAH, NLRC5, C3, and B4GALNT2 SEQ ID SEQ Gene No. Forward sequence (5′to 3′) ID No. Reverse sequence (5′ to 3′) C3 113acaccgcaaggggatattcgggtttg 114 aaaacaaacccgaatatccccttgcg B4GALNT2 115acaccgtgcttttggtcctgagcgtg 116 aaaacacgctcaggaccaaaagcacg (option1)B4GALNT2 117 acaccgtcgatcctcaagatattgag 118 aaaactcaatatcttgaggatcgacg(opition2) GGTA1 119 acaccggggagagaagcagaggatgg 120aaaaccatcctctgcttctctccccg CMAH 121 acaccgtagaaaaggatgaagaaaag 122aaaacttttcttcatccttttctacg NLRC5 123 acaccggcctcagaccccacacagag 124aaaactctgtgtggggtctgaggccg

TABLE 7 Exemplary sequencing primers for px330 plasmids SEQ SEQ IDID No. Forward sequence (5′ to 3′) No. Reverse sequence (5′ to 3′) 125gccttttgctggccttttgctc 126 cgggccatttaccgtaagttatgtaacg

Example 2: Generating a Plasmid Expressing Guide RNA Targeting theRosa26 Locus in Pigs

Pigs with MHC deficiencies will provide transplant grafts that inducelow or no immuno-rejection in a recipient. Exogenous proteins thatinhibit MHC functions will be expressed in pigs to cause MHCdeficiencies. Another goal of ours further along in the project is toinsert one or more exogenous polynucleotides encoding one or moreproteins under the control of a ubiquitous promoter that will directubiquitous expression of the one or more proteins. This example showgenerating a plasmid expressing guide RNA targeting one of suchubiquitous promoter, Rosa26. Rosa26 promoter will direct ubiquitousexpression of a gene at the Rosa26 locus. Thus transgenic pigs will begenerated by inserting transgenes encoding the exogenous proteins at theRosa26 locus, so that the gene product will be expressed in all cells inthe pig. A plasmid expressing guide RNA targeting Rosa26 will be used tofacilitate insertion of a transgene into the Rosa26 locus. This exampleshows exemplary methods for generating plasmids for targeting the Rosa26locus in pigs using the CRISPR/cas9 system. The plasmids were generatedusing the px330 vector, which was be used to simultaneously express aCas9 DNA endonuclease and a guide RNA.

Sequencing Rosa26:

For designing guide RNA targeting Rosa26 locus in a pig, Rosa26 in thepig was sequenced to provide accurate sequence information.

Primer Design: The Rosa26 reference sequence utilized to generateprimers was taken from Kong et. al., Rosa26 Locus SupportsTissue-Specific Promoter Driving Transgene Expression Specifically inPig. PLoS ONE 2014; 9(9):e107945, Li et. al., Rosa26-targeted swinemodels for stable gene over-expression and Cre-mediated lineage tracing.Cell Research 2014; 24(4):501-504, and Li et. al., Identification andcloning of the porcine ROSA26 promoter and its role in transgenesis.Transplantation Technology 2014:2(1). The reference sequence was thenexpanded by searching the pig genome database (NCBI) and by usingEnsembl Genome Browser. The base sequence was separated into four 1218base pair regions to facilitate primer design. Primers were designedusing Integrated DNA Technologies' PrimerQuest Tool and then searchedagainst the Sus scrofa reference genomic sequences using StandardNucleotide BLAST to check for specificity. Primer length was limited to200-250 base pairs. Primer annealing temperature was calculated usingthe New England Tm Calculator for a primer concentration of 1000 nM andthe Taq DNA Polymerase Kit.

PCR: PCR was performed using Taq DNA Polymerase with Standard Taq Buffer(New England Biolabs). DNA template used for the PCR was extracted fromcells isolated from the cloned pig. PCR conditions were 30 cycles of:95° C., 30 seconds; 50° C., 30 seconds, 51° C. 30 seconds, 52° C. 30seconds, 53° C. 30 seconds, 54° C. 30 seconds, 55° C. 30 seconds; and anextension step at 68° for 30 seconds. PCR products were purified usingthe QIAquick PCR Purification Kit (Qiagen). Primers used for sequencingare listed in Table 8.

TABLE 8 Exemplary PCR primers for sequencing Rosa26 SEQ ID No.Primer Name Sequence (from 5′ to 3′) 127 R26F008 tctgattggctgctgaagtc128 R26F013 gtagccagcaagtcatgaaatc 129 R26R013 gggagtattgctgaacctca 130R26F014 tcttgactaccactgcgattg 131 R26R014 gttaggagccagtaatggagtt 132R26F015 agtgtctctgtctccagtatct 133 R26R015 ttggtaaatagcaatcaactcagtg 134R26F016 tttctgctcaagtcacactga 135 R26R016 caagcaatgacaacaacctgata 136R26F017 ttgctttctcctgatcccatag 137 R26R017 cagtgctaatctagagcactacc 138R26F018 cattctcctgaagagctcagaat 139 R26R018 tccattgggctttgtctatactt 140R26F019 gacaaaggaaattagcagagaacc 141 R26R019 aactggtctttcccttggatatt 142R26F020 ctggctgcagcatcaatatc 143 R26R020 gcctctattaattgcctttccc 144R26F021 ccattcacttcgcatccct 145 R26F005 cgggaagtcgggagcata 146 R26R005gaggagaagcggccaatc 147 R26F006 ctgctcttctcttgtcactgatt 148 R26R006gcgggagccactttcac 149 R26F008 tctgattggctgctgaagtc 150 R26R008cgagagcaggtagagctagt 151 R26F010 ggagtgccgcaataccttta 152 R26R010cctggactcatttcccatctc 153 R26F011 gggtggagatgggaaatgag 154 R26F012gctacaccaccaaagtatagca 155 R26R012 tggtggtggaacttatctgattt 156 R26F023agggggtacacattctcctga 157 R26R023 gacctctgggttccattggg 158 R26F024caaagcccaatggaacccag 159 R26F025 gaaggggctttcccaacagt 160 R26F026gcccaagacagggaaaacga 161 R26R026 tgacaactctggtcgctctg 162 R26F028cagagagcctcggctaggta 163 R26R028 aatggctccgtccgtattcc 164 R26F029gggaagtcgggagcatatcg 165 R26R029 cactcccgaggctgtaactg 166 R26F030atggcgtgttttggttggag 167 R26R030 ggagccactttcactgaccc 168 R26F031gggagggtcagtgaaagtgg 169 R26R031 gagggccgtaccaaagacc 170 R26F032ggtcccaaatgagcgaaacc 171 R26R032 gggtccgagagcaggtagag 172 R26F033ccgcctgaaggacgagacta 173 R26R033 cagggcggtccttaggaaaa 174 R26F034gggagtgccgcaataccttt 175 R26R034 gaaattgggctcgtcctcgt 176 R26F035cgaggacgagcccaatttct 177 R26R035 agtgagggggcctaaggttt 178 R26F037actaccactgcgattggacc 179 R26R037 aggagccagtaatggagttgt 180 R26F038cacaactccattactggctcct 181 R26R038 ggagggtagcattccagagg 182 R26F021ccattcacttcgcatccct 183 R26R021 ttgcagatgattgcttcctttc 184 R26F023agggggtacacattctcctga 185 R26R023 gacctctgggttccattggg 186 R26F025gaaggggctttcccaacagt 187 R26R025 gtggcgtatgccccagtatc

Sequencing Analysis: SnapGene software was used to align the DNAsequences. After DNA sequence results were received from the Universityof Minnesota Biogenomics Center, they were uploaded into the SnapGenesoftware and aligned by the software for analysis. Base pairsubstitutions, deletions, and insertions were determined by referencingto the chromatograms and confirmed by comparing sequences of DNAfragments amplified using different primers. When all of the edits andconfirmations were done, the resulting new DNA parent sequence was madeby replacing the original parent DNA sequence with the aligned one (SEQID No. 188, map shown in FIG. 12). The Rosa26 sequence was differentfrom the reference Rosa26 sequence. For example, there were base pairsubstitution, at positions 223, 420, 3927, 4029, and 4066, and base pairdeletion between positions 2692 and 2693. Nucleotide substitutions anddeletions make this sequence unique (FIG. 12). Thus the sequencing dataprovided more accurate sequence information for designing guide RNAtargeting the Rosa26 locus.

Generating the Plasmid Expressing Guide RNA Targeting Rosa26

Oligonucleotides targeting Rosa26 was designed and synthesized by IDT.The sequences of the guide RNA are shown in Table 9. The px330 plasmidexpressing guide RNA targeting Rosa26 was generated using methodsdescribed in Example 1. FIGS. 13A-13E show cloning strategies forcloning the plasmid targeting Rosa 26 (i.e., px330/ROSA exon 1) (FIGS.13A-13E). The constructed px330 plasmid was validated by sequencingusing sequencing primers shown in Table 7.

TABLE 9Exemplary oligonucleotides for making guide RNA constructs targeting Rosa26SEQ ID SEQ ID Gene No. Forward sequence (5′ to 3′) No.Reverse sequence (5′ to 3′) Rosa26 189 acaccgccggggccgcctagagaagg 190aaaaccttctctaggcggccccggcg

Example 3: Inserting HLA-G1 Transgene at Rosa26 Locus in Porcine Cells

A transgene-will be inserted into the Rosa26 locus in pigs so that thepigs will express the transgene in all cells. This example showsexemplary methods for inserting HLA-G1 cDNA into the Rosa26 locus in pigcells (e.g., porcine fetal fibroblasts). The resulting cells will beused to generate pigs expressing HLA-G1 controlled by the Rosa26promoter, which will direct ubiquitous expression of HLA-G1 in the pigs.

The HLA-G1 gene constructs with 1000 bp homology arms specific to theGGTA1 or Rosa26 will be created and verified by PCR and sequencing. ThecDNA sequence of HLA-G1 is shown in Table 2, and the genomic sequence ofHLA-G is shown as SEQ ID: No. 191. The maps of the genomic sequence andcDNA of HLA-G are shown in FIGS. 14A-14B. The flanking regions of theGGTA1 and Rosa26 in the cells will be sequenced. The expression ofHLA-G1 by the construct will be validated. After sequencing andexpression validation, the gene-targeting constructs will be assembledwith the transgene to create a homologous domain repair template thatwill be used to modify somatic pig cells. The CRISPR/Cas technology willbe used to target the GGTA1 or Rosa26 with plasmid-expressed guide RNAoligos, enabling efficient gene targeting and modification. Doublestrand DNA breaks created by guide RNA will be created in the presenceof HLA-G1 gene construct with 1000 bp homology arms inducing DNA repairthat incorporates the transgene. Insertion sites within 50 bp of thepromoter sequence through determined open reading frames (excludingintronic regions) will be tested based on the presence of PAM sequencesand promoter strength to drive transgene expression in the presence ofadditional Cas9 expressing plasmids. The transgenic and knockoutphenotype will be evaluated by flow cytometry (e.g., detection of thetransgenes expression in the cytosol and membrane surface), Westernblotting, and DNA/RNA sequencing.

Example 4: Generating Plasmids that Simultaneously Express Two GuideRNAs

An alternative vector (e.g., px333) simultaneously expressing two guideRNAs will also be used for expressing guide RNA targeting two regions ofa single gene. Targeting two regions of a single gene by CRISPR/cas9system will result in removal of the entire gene between the two cutsites when the DNA is repaired back together. Targeting two regions willincrease the chance of producing a biallelic knockout, resulting inbetter sorts, more biallelic deletions, and overall a higher chance toproduce pigs with a negative genotype, comparing to only targeting onelocus in the gene.

The oligonucleotide pairs used in the px333 plasmid construction willcontain higher G content, lower A content, and as many GGGG quadraplexesas possible, compared with the oligonucleotides used for the px330plasmid. The GGTA1 targets will span nearly the entire GGTA1 gene, whichwill remove the entire gene from the genome. Furthermore, targetingmultiple sites with this strategy will be used when insertingtransgenes, which is another goal of ours further along in the project.

Example 5: Isolating, Culturing and Transfecting Porcine FetalFibroblasts for Making Genetically Modified Pigs

To generate genetically modified pigs using a px330 plasmid expressingguide RNA targeting a gene, the px330 plasmid was transfected intoporcine fetal fibroblasts. The transfected fibroblasts will express theguide RNA that causes disruption of one or more target genes. Theresulting fibroblasts were used for making genetically modified pigs,e.g., by somatic cell nuclear transfer. This example shows isolation andculturing porcine fetal fibroblasts, and transfection of the fibroblastswith a px330 plasmid.

Cell Culture

Fetal fibroblasts cell lines used in the generation of geneticallymodified pigs included: Karoline Fetal (derived from female porcineponor P1101, which provided a high islet yield after islet isolation),Marie Louise Fetal (derived from female porcine donor P1102, whichprovided a high islet yield after islet isolation), Slaughterhouse pig#41 (Male; showed a high number of islets in the native pancreas (asassessed by a very high dithizone (DTZ) score)), Slaughterhouse pig #53(showed a high number of islets in the native pancreas as assessed by ahigh dithizone (DTZ) score).

Muscle and skin tissue samples taken from each of these pigs weredissected and cultured to grow out the fibroblast cells. The cells werethen harvested and used for somatic cell nuclear transfer (SCNT) toproduce clones. Multiple fetuses (up to 8) were harvested on day 30.Fetuses were separately dissected and plated on 150 mm dishes to growout the fetal fibroblast cells. Throughout culture, fetus cell lineswere kept separate and labeled with the fetus number on each tube orculture vessel. When confluent, cells were harvested and frozen back atabout 1 million cells/mL in FBS with 10% DMSO for liquid nitrogencryo-storage.

Culture medium preparation: 5 mL Glutamax, 5 mL pen/strep, and 25 mLHI-FBS (for standard 5% FBS medium; use 10% FBS for sorted cells) wereadded to a 500 mL bottle of DMEM, high glucose, no glutamine, no phenolred. Centrifuge settings for spinning down all fetal fibroblasts were 5minutes at 0.4 rcf (1600 rpm) at 4° C. Cells were thawed from liquidnitrogen storage by warming quickly to 37° C. in water bath. The thawedcells were quickly transferred to about 25 mL fresh, pre-warmed culturemedium (enough to dilute the DMSO sufficiently). The cells were thenspun down, the supernatant was removed and the cells were re-suspendedin 1-5 mL fresh culture medium for counting or plating. Cells received amedium change every 3-4 days with pre-warmed medium, and were passagedwhen 90-100% confluent using TrypLE Express Dissociation Reagent.

Harvesting Adherent Fibroblasts: The medium was aspirated off the cells.DPBS was added to wash the cells. Pre-warmed (37° C.) TrypLE Expressreagent was added to the cells. Minimum amount of the reagent was usedto cover the cell layer thinly. The cells were incubated at 37° C. for10 minutes. A volume of culture medium containing FBS was added to theTrypLE cell suspension to neutralize the enzyme. The cell suspension waspipetted up and down to dislodge all cells from the culture surface. Thecell suspension was transferred to a 15 or 50 mL conical tube on ice.The plate/flask was checked under a microscope to ensure all cells werecollected. Sometimes a medium wash helped collect cells that were leftbehind. The cells were spun down, and then re-suspended with freshculture medium (between 1-5 mL for counting). If counting, a 1:5dilution of the cells suspension was prepared by adding 20 μL cellsuspension to 80 μL 0.2% Trypan Blue. The suspension was mixed well bypipetting up and down. 12-14 μL of the dilution was added to ahemocytometer to count the 4 corners. The numbers were averaged. Forexample, counting 20, 24, 22, 22 for each corner yielded an average of22. This number was multiplied by the dilution factor 5, yielding110×10⁴ cells/mL. The number was adjusted to 10⁶ by moving the decimaltwo places to the left, 1.10×10⁶ cells/mL. Finally, the numbers weremultiplied by how many mL's the original sample was taken from to getthe total number of cells.

Transfection of Fetal Fibroblasts

This experiment was to transfect fetal fibroblasts. The transfectedfetal fibroblasts were used to generate genetically modified animalusing the somatic cell nuclear transfer technique.

The GFP plasmid used (pSpCas9(BB)-2A-GFP) for transfection was an exactcopy of the px330 plasmid, except that it contained a GFP expressionregion.

GFP transfected control cells: Transfections were done using the NeonTransfection System from Invitrogen. Kits came in 10 μL and 100 μL tipsizes. A day or two before the experiment, cells were plated inappropriate culture vessel depending on size of experiment and desiredcell number and density. About 80% confluence was achieved on day oftransfection.

On the day of the experiment, Neon module and pipette stand was set upin a biohood. A Neon tube was placed in the pipette stand and 3 mL ofBuffer E (Neon Kit) was added to the Neon tube. The module was turned onand adjusted to desired settings (for fetal porcine fibroblasts: 1300 V,30 ms, 1 pulse). Cells were harvested using TrypLE and counted todetermine the experimental setup. Needed amount of cells weretransferred to a new tube and remaining cells were re-plated. Cells werespun down after counting, and re-suspended in PBS to wash. The cellswere spun down and re-suspended in Buffer R (Neon Kit) according toTable 10 for the number of cells and tip sizes.

TABLE 10 Exemplary Neon ® plate formats, volumes, and recommended kitsVol. DNA siRNA plating Buffer R or Format Cell Type (μg) (nM) Neon ®Tipmedium Cell no. Buffer T 96-well Adherent  0.25-0.5 10-200 10 μL 100 μL1-2 × 10⁴ 10 μL/well Suspension 0.5-1 10 μL 2-5 × 10⁴ 10 μL/well 48-wellAdherent 0.25-1  10-200 10 μL 250 μL 2.5-5 × 10⁴  10 μL/well Suspension0.5-2 10 μL 5-12.5 × 10⁴   10 μL/well 24-well Adherent 0.5-2 10-200 10μL 500 μL 0.5-1 × 10⁵  10 μL/well Suspension 0.5-3 10 μL 1-2.5 × 10⁵  10μL/well 12-well Adherent 0.5-3 10-200 10 μL 1 mL 1-2 × 10⁵ 10 μL/wellSuspension 0.5-3 10 μL 2-5 × 10⁵ 10 μL/well  6-well Adherent 0.5-3 (10μL)  10-200 10 μL/100 μL 2 mL 2-4 × 10⁵ 10 μL or 5-30 (100 μL) 100μL/well Suspension 0.5-3 (10 μL)  10 μL/100 μL 0.4-1 × 10⁶  10 μL or5-30 (100 μL) 100 μL/well 60 mm Adherent   5-30 10-200 100 μL 5 mL 0.5-1× 10⁶  100 μL/well  Suspension   5-30 100 μL 1-2.5 × 10⁶  100 μL/well 10 cm  Adherent   5-30 10-200 100 μL 10 mL 1-2 × 10⁶ 100 μL/well Suspension   5-30 100 μL 2-5 × 10⁶ 100 μL/well 

Appropriate amount of DNA according to Table 10 was added to cellsuspension and mixed by pipetting up and down. A Neon tip was appliedfrom the kit to the Neon pipette to aspirate the volume of cellsuspension into the Neon tip. The pipette was placed into the Neon tubein the pipette stand so that the Neon tip was submerged in the Buffer E.START was pressed on module interface until a “complete” messageappeared. The pipette was removed from the pipette stand to eject thecell suspension into a volume of pre-warmed culture medium withoutantibiotics in a well of appropriate size according to Table 10.

The above steps were repeated until the entire cell suspension was used.Neon tips were changed every 2 transfections, and Neon tubes werechanged every 10 transfections. The cells were incubated at 37° C. for24 hours, and then the medium was changed with normal culture mediumcontaining antibiotics. The resulting cells were cultured for about 5days to allow for Cas9 cleavage, complete recycling of surface proteinsafter gene knockout, and proper cell division before sorting. The cellstransfected with the GFP plasmid were shown in FIG. 15.

Example 6: Verifying Guide RNA Production by Px330 Plasmids Using RNAPolymerase

After a px330 plasmid is transfected to porcine fetal fibroblasts, theexpression of guide RNA by the px330 plasmid will be verified using anRNA polymerase.

The guide RNA production by px330 plasmids will be verified by in vitrotranscription of the correctly constructed plasmids by an RNApolymerase. The experiment will use the T7 RNA polymerase with apromoter introduced through PCR of the target region. Production ofsgRNA by the T7 RNA polymerase will indicate that the plasmid istranscribed and the sgRNA is present in the cells. Gel verification ofthe reaction product (e.g., sgRNA) size will be used to confirm sgRNAtranscription by the RNA polymerase.

Example 7: Fluorescence In Situ Hybridization (FISH) to the GGTA1 Gene

Gene disruption by CRISPR/cas9 was verified using FISH in a cell. Thisexample shows exemplary methods for detecting GGTA1 gene usingfluorescence in situ hybridization (FISH). The methods here were used toverify the presence or absence of a GGTA1 gene in a cell from an animal(e.g., an animal with GGTA1 knocked out).

Preparation of FISH Probes:

GGTA1 DNA was extracted from an RP-44 pig BAC clone (RP44-324B21) usingan Invitrogen PureLink kit. The DNA was labeled by nick translationreaction (Nick Translation Kit—Abbott Molecular) using Orange—552 dUTP(Enzo Life Science). Sizes of the nick translated fragments were checkedby electrophoresis on a 1% TBE gel. The labeled DNA was precipitated inCOT-1 DNA, salmon sperm DNA, sodium acetate and 95% ethanol, then driedand re-suspended in 50% formamide hybridization buffer.

Hybridization of FISH Probes:

The probe/hybridization buffer mix and cytogenetic slides from pigfibroblasts (15AS27) were denatured. The probe was applied to theslides, and the slides were hybridized for 24 hours at 37° C. in ahumidified chamber. The probe used is shown as SEQ ID No: 192.

FISH Detection, Visualization and Image Capture:

After hybridization, the FISH slides were washed in a 2×SSC solution at72° C. for 15 seconds, and counterstained with DAPI stain. Fluorescentsignals were visualized on an Olympus BX61 microscope workstation(Applied Spectral Imaging, Vista, Calif.) with DAPI and FITC filtersets. FISH images were captured using an interferometer-based CCD cooledcamera (ASI) and FISHView ASI software. The FISH image is shown in FIG.16.

Example 8: Phenotypic Selection of Cells with Cas9/Guide RNA-MediatedGGTA1 Knockout

Disruption of GGTA1 gene by the Cas9/guide RNA system were verified bylabeling GGTA1 gene products. The GGTA1 knockout will be used as amarker for phenotypic sorting in knockout experiments. The GGTA1 geneencoded for a glycoprotein found on the surface of pig cells that if hadbeen knocked out, would result in the glycoprotein being absent on thecell's surface. The lectin used to sort for GGTA1 negative cells wasIsolectin GS-IB₄ Biotin-XX conjugate, which selectively bound terminalalpha-D-galactosyl residues, such as the glycoprotein produced by theGGTA1 gene.

Porcine fetal fibroblast cells were transfected with px330 plasmidexpressing guide RNA targeting GGTA1 (generated in Example 1).

To select for negative cell after transfection, the cells were allowedto grow for about 5 days to recycle their surface proteins. The cellswere then harvested, and labeled with the IB₄ lectin. The cells werethen coated with DynaBeads Biotin-Binder, which were 2.8 micronsupermagnetic beads that had a streptavidin tail that bound very tightlywith the biotin-conjugated lectin on the surface of the cells. Whenplaced in a magnet, the “positive” cells with lectin/beads bound on thesurface stick to the sides of the tube, while the “negative” cells didnot bind any beads and remained floating in suspension for an easyseparation.

In detail, the cells were harvested from a plate using a TrypLE protocoland collected into a single tube. The cells were spun down, andre-suspended in 1 mL of sorting medium (DMEM, no supplements) to count.If less than 10 million cells, the cells were spun down and thesupernatant was discarded. In a separate tube, IB₄ lectin (1 μg/μL) wasdiluted by 5 μL to 1 mL of sorting medium (final concentration 5 μg/mL).The cell pellet was re-suspended with the 1 mL of diluted lectin. Thecell suspension was incubated on ice for about 15-20 minutes, withgentle sloshing every few minutes.

Biotin beads were prepared during incubation. A bottle of beads werevortexed for 30 seconds. 20 μL beads/1M cells were added to 5 mL ofsorting medium in a 15 mL conical tube. The tube was vortexed, placed inDynaMag-15 magnet and let stand for 3 minutes. Medium were removed. 1 mLof fresh sorting medium was added and the tube was vortexed to wash thebeads. The washed beads were placed on ice until use.

After cell incubation, cell suspension's volume was brought to 15 mLwith sorting medium to dilute the lectin. The cells were spun andre-suspended with 1 mL of the washed biotin beads. The suspension wasincubated on ice for 30 minutes in a shaking incubator at 125 rpm. Thecell suspension was removed from shaking incubator and inspected. Smallaggregates might be observed.

5 mL of sorting medium was added to the cell suspension and the tube wasplaced in the DynaMag-15 for 3 minutes. The first fraction of“negatives” cells was collected and transferred to a new 15 mL conicaltube. Another 5 mL sorting medium was added to wash the “positive” tubethat was still on the magnet. The magnet was inverted several times tomix the cell suspension again. The tube was let stand for 3 minutes toseparate cells. The second “negative” fraction was then removed andcombined with the first fraction. 10 mL sorting medium was added to the“positive” tube. The tube was removed from the magnet, and placed in anice bath until ready to use.

The tube of “negative” fractions was placed onto the magnet to provide asecondary separation and remove any bead-bound cells that might havecrossed over from the first tube. The tube was kept on the magnet for 3minutes. The cells were pipetted away from the magnet and transferredinto a new 15 mL conical tube. The original “positive” tube and thedouble sorted “negative” tube were balanced and cells in them were spundown. The pellet of the “positives” appeared a dark, rusty red. The“negative” pellet was not visible, or appeared white.

Each pellet was re-suspended in 1 mL of fresh culture medium (10% FBS)and plated into separate wells on a 24-well plate. The wells wereinspected under a microscope and diluted to more wells if necessary. Thecells were cultured at 37° C. The genetically modified cells, i.e.,unlabeled cells were negatively selected by the magnet (FIG. 17A). Thenon-genetically modified cells, i.e., the labeled cells had accumulatedferrous beads on the cell surface (FIG. 17B).

Example 9: Making GGTA1/CMAH/NLRC5 Triple Knockout Pigs

This example shows exemplary methods for generating a triple knockoutpigs. A triple knockout pig can have reduced protein expression of threeof the following: NLRC5, TAP1, C3, CXCL10, MICA, MICB, CIITA, CMAH,GGTA1 and/or B4GALNT2. One of such triple knockout pig wasGGTA1/CMAH/NLRC5 triple knockout pigs using CRISPR/cas9 system. The pigsprovided islets for transplantation. Porcine islets with disruptedGGTA1/CMAH/NLRC5 had MHC class I deficiency and will induce low or noimmuno-rejection when transplanted to a recipient.

Transfection of Fetal Fibroblasts

The px330 plasmids expressing guide RNA targeting GGTA1, CMAH, and NLRC5generated in Example 1 were transected in porcine fetal fibroblasts. Pigfetal fibroblasts were cultured in DMEM containing 5-10% serum,glutamine and penicillin/streptomycin. The fibroblasts wereco-transfected with two or three plasmids expressing Cas9 and sgRNAtargeting the GGTA1, CMAH or NLRC5 genes using Lipofectamine 3000 system(Life Technologies, Grand Island, N.Y.) according to the manufacturer'sinstructions.

Counter-Selection of GGTA1 KO Cells

Four days after transfection, the transfected cells were harvested andlabeled with isolectin B4 (IB4)-biotin. Cells expressing αGal werelabeled with biotin conjugated IB4 and depleted by streptavidin coatedDynabeads (Life Technologies) in a magnetic field. The αGal deficientcells were selected from the supernatant. The cells were examined bymicroscopy. The cells containing no or very few bound beads aftersorting were identified as negative cells.

DNA Sequencing Analysis of the CRISPR/Cas9 Targeted GGTA1, CMAH andNLRC5 Genes

Genomic DNA from the IB4 counter-selected cells and cloned pig fetuseswere extracted using Qiagen DNeasy Miniprep Kit. PCR was performed withGGTA1, CMAH and NLRC5 specific primer pairs as shown in Table 11. DNApolymerase, dNTPack (New England Biolabs) was used and PCR conditionsfor GGTA1 were based on annealing and melting temperature ideal forthose primers. The PCR products were separated on 1% agarose gel,purified by Qiagen Gel Extraction Kit and sequenced by the Sanger method(DNA Sequencing Core Facility, University of Minnesota) with thespecific sequencing primers as shown in Table 7. FIGS. 18A-18C show thesequences and agarose gel images of the PCR products.

TABLE 11Exemplary PCR primers for amplifying genomic DNA from genetically modifiedcells and animals SEQ ID SEQ Gene No. Forward sequence (5′ to 3′) ID No.Reverse sequence (5′ to 3′) GGTA1 193 cttcgtgaaaccgctgtttatt 194gactggaggactttgtcttctt CMAH 195 tgagttccttacgtggaatgtg 196tcttcaggagatctgggttct NLRC5 197 ctgctctgcaaacactcaga 198tcagcagcagtacctcca

Somatic Cell Nuclear Transfer (SCNT)

SCNT was performed as described by Whitworth et al. Biology ofReproduction 91(3):78, 1-13, (2014), which is incorporated herein byreference in its entirety. The SCNT was performed using in vitro maturedoocytes (DeSoto Biosciences Inc., St. Seymour, Tenn.). Cumulus cellswere removed from the oocytes by pipetting in 0.1% hyaluronidase. Onlyoocytes with normal morphology and a visible polar body were selectedfor SCNT. Oocytes were incubated in manipulation media (Ca-free NCSU-23with 5% FBS) containing 5 μg/mL bisbenzimide and 7.5 μg/mL cytochalasinB for 15 min. Oocytes were enucleated by removing the first polar bodyplus metaphase II plate. A single cell was injected into each enucleatedoocyte, fused, and activated simultaneously by two DC pulses of 180 Vfor 50 μsec (BTX cell electroporator, Harvard Apparatus, Hollison,Mass., USA) in 280 mM Mannitol, 0.1 mM CaCl₂, and 0.05 mM MgCl₂.Activated embryos were placed back in NCSU-23 medium with 0.4% bovineserum albumin (BSA) and cultured at 38.5° C., 5% CO₂ in a humidifiedatmosphere for less than 1 hour, and transferred into the surrogatepigs.

Example 10: Making NLRC5 Knockout Non-Human Animals Expressing an ICP47Transgene

This example shows exemplary methods for generating genetically modifiednon-human animals (e.g., pigs) with reduced expression of one or moreendogenous genes and meanwhile expressing one or more transgenes. Suchgenerating genetically modified non-human animals (e.g., pigs) will havereduced expression of one or more of NLRC5, TAP1, C3, CXCL10, MICA,MICB, CIITA, CMAH, GGTA1 and/or B4GALNT2, and meanwhile expressing oneor more ICP47, CD46, CD55, CD59 HLA-E, HLA-G (e.g., HLA-G1, HLA-G2,HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), L2, Spi9, galectin-9 CD47,B2M, PD-L1, and/or PD-L2. One of such animal will have disrupted NLRC5gene and meanwhile overexpressing a transgene encoding ICP47. NLRC5disruption and ICP47 expression will suppress MHC-1 assembly andfunction. Thus, cells, tissues, and/or organs from the geneticallymodified non-human animals (e.g., pigs) will induce low or noimmuno-rejection when transplanted to a recipient.

Cloning Rosa26 Promoter

The Rosa26 promoter sequence will be obtained by searching genomedatabases (NCBI) using mouse Rosa26 promoter sequence and human Rosa26promoter sequence as references. The non-human animal's version of theRosa26 will be obtained. Primers will be designed to amplify a DNAfragment harboring potential Rosa26 promoter (e.g., porcine Rosa26promoter) by PCR using the non-human animal's (e.g., a domestic pig's)genomic DNA as a template. AscI and MluI sites will be added to the 5′of forward and reverse primers, respectively. Pwo SuperYield DNAPolymerase (Roche, Indianapolis, Ind.) will be used and PCR conditionswill be as follows: 94° C., 2 minutes; 94° C., 15 seconds, 55° C., 30seconds; 72° C. 4 minutes for 15 cycles; 94° C., 15 seconds, 55° C., 30seconds; 72° C. 4 minutes and 5 seconds added to each cycle for 25cycles; and a final extension step of 72° C. for 8 minutes. The PCRproduct will be subsequently cloned into pCR-XL-TOPO vector (Invitrogen,Carlsbad, Calif.) to generate pCR-XL-Rosa26. The non-human animal's Rosapromoter (e.g., porcine Rosa26 promoter) will be sequenced usingdesigned primers.

Construction of Transgenic Vector

CMV promoter and multiple cloning site (MCS) of pEGFP-N1 (Clontech, PaloAlto, Calif.) will be replaced by a linkerAseI-NruI-AscI-SalI-MluI-PvuI-BamHI. A 3.9 kb fragment containing thepotential non-human animal's (e.g., porcine) Rosa26 promoter will beexcised from pCR-XL-Rosa26 with AscI and MluI digestion and inserted tothe promoterless pEGFP-N1 between AscI and Mlu I sites, resulting inplasmid pRosa26-EGFP. Human ICP47 cDNA will be cloned to replace EGFP inthe plasmid. The control vector will be constructed by cloning of ICP47cDNA to the downstream of murine MHC class I H-2K^(b) promoter at EcoRIsite, resulting in plasmid pH-2K^(b)-ICP47.

Transient Transfection

NLRC5 KO fetal fibroblast cells from the NLRC5 knockout non-humananimals (e.g., pigs) made by the methods of Example 1 or Example 2 willbe obtained. To compare promoter strength among Rosa26, H-2K^(b), andCMV, fetal fibroblast cells will be transfected with pRosa26-ICP47,pH-2K^(b)-ICP47 and pEGFP-N1 by using Neon™ Transfection System(Invitrogen, Carlsbad, Calif.) as per the manufacturer instructions.3×10⁵ cells will be mixed with 1.5 μg of each DNA, respectively, andelectroporated at 1300V, 30 ms, 1 pulse. Then cells will be cultured at37° C. with 5% CO₂ and 10% O₂. After 48 hours, cells will be harvestedand ICP47 expression will be examined by Western blot and/or flowcytometry. Untransfected fetal fibroblasts will be used as a control.

Establishment of EGFP Stable Cell Line

NLRC5 KO fetal fibroblasts at 80-90% confluence will be harvested withtrypsin and washed with calcium and magnesium free DPBS (Invitrogen,Carlsbad, Calif.). pRosa26-ICP47 will be linearized by Asc I digestion.Transfection will be performed by using Neon™ Transfection System(Invitrogen). Briefly, 10⁶ cells will be suspended in 120 μl of R bufferand 2 μg of linearized DNA will be added. Cells will be electroporatedat 1300V, 30 ms, 1 pulse, and plated onto collagen I coated plates (BD)in the culture media without antibiotics. After 48 hours, the culturemedia will be replaced with selection media containing 100 μg/ml of G418(Invitrogen). After 10 days of G418 selection the ICP47 positive cellswill be isolated by flow sorting. The selected cells will be expandedand the second flow sorting will be performed to purify and enrich theICP47 positive cells.

Somatic Cell Nuclear Transfer

SCNT will be performed using in vitro matured oocytes (DeSotoBiosciences Inc. St Seymour, Tenn. and Minitub of America, Mount Horeb,Wis.). Cumulus cells will be removed from the oocytes by pipetting in0.1% hyaluronidase. Only oocytes with normal morphology and a visiblepolar body will be selected for cloning. Oocytes will be incubated inmanipulation media (Ca-free NCSU-23 with 5% FBS) containing 5 mg/mLbisbenzimide and 7.5 mg/mL cytochalasin B for 15 min. Following thisincubation period, oocytes will be enucleated by removing the firstpolar body and metaphase II plate, and one single cell will be injectedinto each enucleated oocyte. Electrical fusion will be induced with aBTX cell electroporator (Harvard Apparatus, Holliston, Mass.). Coupleswill be exposed to two DC pulses of 140 V for 50 ms in 280 mM Mannitol,0.001 mM CaCl₂, and 0.05 mM MgCl₂. One hour later, reconstructed oocyteswill be activated by two DC pulses of 120 V for 60 ms in 280 mMMannitol, 0.1 mM CaCl₂, and 0.05 mM MgCl₂. After activation, oocyteswill be placed back in NCSU-23 medium with 0.4% bovine serum albumin BSAand cultured at 38.5° C., 5% CO₂ in a humidified atmosphere, for lessthan 1 h before being transferred into the recipient. Recipients will besynchronized non-human animals (e.g., occidental pigs) on their firstday of estrus.

Genotyping of ICP47 Transgenic Fetuses

The pregnancy will be terminated at day 35 and fetuses will beharvested. Genomic DNA will be extracted using DNeasy Blood & Tissue Kit(Qiagen). PCR primers will be designed to detect ICP47 cDNA sequence inthe genome. The 20 μl of reaction mixture contained 10 μl of 2× Go-TaqGreen Master Mix (Promega, Madison, Wis.), 5 pmol of each primer, and 50ng of genomic DNA. PCR will be performed to detect the presence orabsence of ICP47 insert. Genomic DNA extracted from normal cells will beused as negative control.

Example 11: Making NLRC5 Knockout Non-Human Animals by Injecting RNAEncoding Cas9 and Guide RNA

An alternative approach to targeting a gene using CRISPR/cas will be todirectly inject an RNA molecule encoding Cas9 and a guide RNA (e.g.,single guide RNA (sgRNA)) into a cell to disrupt a gene by CRISPR/cas9system. This example shows exemplary methods for disrupting NLRC5 innon-human animals (e.g., pigs) by injection of RNA encoding Cas9 and asingle guide RNA (sgRNA).

The sgRNA targeting the NLRC gene will be designed and synthesized. Toconstruct the Cas9 encoding plasmid, the Cas9 coding sequence will besynthesized and then cloned into the pEASY-T1vector, which harbors a T7promoter for the in vitro transcription of Cas9. A SV40 polyadenylationsignal will be at the 3′ end of the Cas9 cassette, and a unique HindIIIrestriction site will be outside of SV40 signal for linearization. TheT7 promoter containing sgRNA scaffold will also be ordered and clonedinto a promoterless pUC19 vector. Two BsaI restriction sites will beused for the spacer insertion and the plasmid will be linearized by PsiIfor in vitro transcription. For targeting vector construction, asite-specific 20 nt spacer will be synthesis and cloned into T7-sgRNAscaffold between BsaI restriction sites.

To prepare Cas9 mRNA, T7-Cas9 expression plasmid will be linearized byHindIII, and purified using DNA Clean & Concentrator™-5 (ZYMO Research).

To prepare sgRNA, the sgRNA vector will be linearized using PsiI andpurified by using DNA Clean & Concentrator™-5 (ZYMO Research).

All the linearized plasmid will be in vitro transcribed by T7 High YieldRNA Synthesis Kit (NEB) following the manufacturer's instruction. Tosynthesize Cas9 mRNA, the m7G(5′)G RNA Cap Structure Analog (NEB) willbe additionally added to stabilized the transcribed mRNA. Prepared RNAwill be purified using MicroElute RNA Clean-Up Kit (Omega) and recoveredin DEPC water.

The zygotes from the non-human animal, e.g., Bama minipig, will becollected on the next of insemination, transferred to manipulationmedium and subjected to a single cytoplasmic microinjection of 2-10 plof 125 ng/μl Cas9 mRNA and 12.5 ng/μl sgRNA. Alternatively, fertilizedoocytes of the non-human animal will be collected. The Cas9 and sgRNAwill be injected into the fertilized oocytes to generate geneticallymodified offspring. To test the viability of non-human animal (e.g.,pigs) embryos after RNA injection, in vitro produced parthenogeneticembryos will be used for preliminary experiment. For parthenogeneticembryos preparation, non-human animal (e.g., pig) ovaries will becollected, washed with pre-warmed saline and follicles aspirated.Oocytes will be washed in TL-HEPES before culturing in maturation mediumfor 44 hours. Matured MII oocytes will be depleted off surroundingcumulus cells by gentle pipetting, followed by electrical activation bytwo direct current pulses (1-sec interval) of 1.2 kV/cm for 30microseconds. Activated oocytes will be transferred to TL-HEPES mediumand subjected to a single 2-10 pl cytoplasmic injection of 125 ng/μlCas9 mRNA and 12.5 ng/μl sgRNA. Zygotes and activated oocytes will becultured to blastocyst stage in PZM3 medium for 144 hours under 5% CO2,39° C.

Cas9 mRNA and the guide RNA will be co-injected to non-human animalzygotes (e.g., pig zygotes). The in vitro developmental efficiencies ofzygotes injected with Cas9 mRNA/guide RNA and zygotes injected withwater will be measured to determine effect of microinjectionmanipulation the Cas9 mRNA/guide RNA on non-human animal (e.g., pig)early embryonic development.

The injected embryos will be transferred into surrogate non-humananimals (e.g., pigs) to produce offspring (e.g., piglets). The survivedembryos will be transferred into the oviduct of recipient gilts on theday or 1 day after estrus, following mid-line laparotomy under generalanesthesia. Pregnancy will be diagnosed about after 28 days, and thenchecked regularly at 2-week intervals by ultrasound examination. All ofthe microinjected offspring (e.g., piglets) will be delivered by naturalbirth.

A total of 76 injected embryos will be transplanted into 5 surrogatemothers in 5 independent experiments. Insertions or deletions in thetargeting sites of the NLRC5 gene will be detected by T7 endonuclease I(T7EI) assay. Genotypes of the offspring (e.g., piglets) will beanalyzed by Sanger sequencing of the PCR products containing thetargeting site of each individual offspring (e.g., piglets).

Example 12: Identifying Immune Cells that Respond to Porcine IsletXenografts in Nonhuman Primates

This example shows exemplary methods for identifying the targetingimmune cells for immune intervention in cellular xenotransplantation. Tothis end, the phenotypes of circulating and graft T and B lymphocytesubsets with effector and regulatory functions were studied incynomolgus macaques (CM) receiving immunosuppression without and withdonor antigen-specific immunotherapy for the prevention of porcine isletxenograft rejection.

Cellular immunity to intraportal porcine islet xenografts wasretrospectively analyzed in 4 cohorts of diabetic CM: induction withα-CD40 and maintenance with CTLA4-Ig and rapamycin (Cohort A; n=4; graftfunction 77 to 333 days); induction with CTLA4-Ig and maintenance withα-CD40 and rapamycin (Cohort B; n=3; stable graft function more than 180days); induction with α-CD40, α-CD20, and rapamycin, no maintenance(Cohort C; n=2; graft function for 32 and 40 days), and induction withperitransplant infusions of apoptotic donor leukocytes under the coverof α-CD40, α-CD20, and rapamycin, no maintenance (Cohort D; n=3; graftfunction for 81, 100, and 113 days). The frequencies of circulatingimmune cell subsets and liver mononuclear cells (LMNC) at sacrifice weredetermined by flow cytometry. LMNC were also analyzed for effectormolecules after ex-vivo stimulation with donor antigen. Statisticalsignificance was determined using unpaired t test with or withoutWelch's correction.

Baseline frequencies of circulating immune cell subsets were notdifferent between Cohorts. Compared with Cohort A CM, through day 100post-transplant, Cohort B CM showed: i) significant increases in ratiosof naïve (CD3−CD20+CD21+CD27−) vs. activated memory(CD3−CD20+CD21+CD27+) and immature (CD3−CD19+CD27−IgM+) vs. mature(CD3−CD19+CD27+CD38+) circulating B cells, ii) significant increases incirculating Bregs (CD19+CD24hiCD38hi), Tregs (CD4+CD25+FoxP3+CD127), andNatural Suppressor Cells (NSC; CD122+CD8+), and iii) comparablecirculating frequencies of CD8+ effector memory (TEM) cells(CD2hiCD28-CD8+). Cohort C but not D CM showed a significant expansionof CD8+ TEM at day 14 post-transplant. Compared with Cohort C CM, at day50±10 post-transplant, Cohort D CM showed significant increases incirculating frequencies of Tregs and NSC. By day 50, there was also asignificant expansion of CD8+ TEM in Cohort D. In CM terminated becauseof presumed rejection, LMNC showed a substantial presence of CXCR3+CD4+and CD8+ T cells and CD20+B cells (including in α-CD20 treated Cohort Cand D CM); CD8+ TEM were the predominant phenotype among LMNCs. Uponex-vivo stimulation with donor antigen, these CD8+ TEM showed abundantstaining for IFN-γ, TNF-α, and Perforin.

The results provided insights into the effects of immunotherapy oncellular immunity in pig-to-CM islet xenotransplantation and identify Bcells and CD8+ TEM as targets for immune intervention in cellularxenotransplantation.

Example 13: Suppressing SLA on Pig Islets Inhibited Human CD8+ T CellsResponse to the Pig Islets

To determine whether suppression of MHC in pig islets (e.g., SLA) caninhibit T cell activation in a human recipient, SLA antibodies were usedto suppress MHC on the pig islets, and human T cells' response to thepig islets was examined.

Human peripheral blood mononuclear cells were cultured with adult pigislets for 7 days with or without an anti-SLA class I blocking antibody.Proliferation of highly purified human CD8+ T cells (hCD8), human CD4+ Tcells (hCD4), and human natural killer cells (hNK) were measured. Theproliferation of the highly purified human CD8+ T cells, but not CD4+Tor NK cells, was inhibited. The recognition of MHC class I molecules onpig islets was blocked by the anti-SLA class I blocking antibody after 7days in the mixed culture (FIG. 19A).

Adult pig islets cultured with or without highly purified lymphocytesfor 7 days in the present or absence of an anti-SLA class I blockingantibody. The viability of the cultured cells was assessed by acridineorange (AO) and propidium iodide (PI) staining. Cytotoxicity of purifiedCD8+ T cells was inhibited in the presence of the anti-SLA I antibody(FIG. 19B). In spite of significant proliferation, CD4+ T cells leftislets relatively unharmed when compared to the cytotoxicity of CD8+ Tcells (FIG. 19B).

Example 14: Suppressing T Cell Activation by ECDI-Fixed Splenocytes in aMonkey Transplanted with Porcine Islets

To determine whether apoptotic splenocytes from a xenograft donor cansuppress immuno-rejection of the xenograft by a recipient, the apoptoticsplenocytes from the donor were administered to the recipient before andafter transplant. Then T cell activation in the recipient's PBMCs wasexamined.

Porcine islets were transplanted to a diabetic monkey. Apoptoticsplenocytes prepared from islets donor were administered to the monkey 1day before and 7 days after transplant. PBMCs were collected from themonkey before transplantation, and 7, 14, 28, 49, 77, and 91 days aftertransplantation. Direct and indirect T cell activation in the PBMCs wasexamined by ELISPOT. The ELISPOT result was shown as spot-forming cells(SFC)/10⁶ PBMCs (FIG. 20A). On day 141, an islet was collected from themonkey and CD8 was detected by immunohistochemistry using anti-CD8antibody (FIG. 20B). PBMCs from 42 non-transplanted monkeys were used asa negative control (“Controls”). PBMCs from 10 monkeys transplanted withnon-genetically modified porcine islets were used as a positive control(“Rejectors”). Administration of splenocytes significantly reduced Tcell activation induced by the porcine islets in the monkey.

Example 15: Treating Diabetes by Transplanting Immuno-Modulated PorcineIslets and ECDI-Fixed Splenocytes from the Same Donor in Monkeys withoutMaintenance of Immunosuppression

In addition to testing the immunosuppression effect of ECDI-fixed donorcells on immune cells in Example 14, experiments in this exampleexamined the immunosuppression effect of ECDI-fixed donor cells in vivo(in monkeys). The results showed that ECDI-fixed splenocytes from a pigreduced the immuno-rejection in a monkey transplanted with islets fromthe pig.

A diabetic monkey was transplanted with porcine islets. The monkey wasgiven ECDI-fixed donor splenocytes (by intravenous infusion) 7 daysbefore and 1 day after the transplantation. Immunosuppression drugs weregiven from the day of transplantation through day 21 after thetransplantation. Small doses of exogenous insulin were administeredthrough day 21 after the transplantation. The exogenous insulin (shownin gray bars) needed to maintain normal blood glucose level was reducedon the day of transplantation and completely stopped on day 21. Bloodglucose level (shown in lines) became normal immediately aftertransplantation and continued to be normal despite discontinuation ofinsulin on day 21. The blood glucose level kept normal without exogenousinsulin over day 100 after transplantation (FIG. 21A). The bloodC-peptide levels including the peak value after transplantation, therandom level, and the level under fasting and glucose-stimulationconditions were tested (FIG. 21B).

The glucose metabolism of the monkey was examined by intravenous glucosetolerance test (IVGTT) (FIGS. 21C and 21D). In IVGTT, exogenous glucosewas injected to the monkey, and the blood glucose level was measuredover time after the injection. IVGTT was performed on the monkey on day28 and day 90 after transplantation. Non-transplanted monkeys treatedwith or without streptozotocin were used as controls. Thenon-transplanted monkeys treated with streptozotocin were used as adiabetic control. The blood glucose (FIG. 21C) and C-peptide (FIG. 21D)levels were measured and compared with the controls.

Example 16: Tolerizing a Recipient and Transplantation with ECDI-FixedCells

Cells from a transplant donor will be fixed by ECDI and used to suppressimmuno-rejection in a recipient. This example shows exemplary methodsfor tolerizing a transplant recipient with ECDI-fixed geneticallymodified cells. Human recipients in need of transplantation will betreated with ECDI fixed cells to tolerize the recipient totransplantation. The ECDI fixed cells will be genetically modified, forexample, GGTA1 and CMAH will be knocked out. B4GALNT2 will also beknocked in some of the ECDI fixed cells. Some or all of the ECDI fixedcells will also express one or more genes that are ICP47, CD46, CD55, orCD59.

The ECDI fixed cells will be given to the recipient about 7 days beforetransplantation and again at about 1 day after transplantation.

A dose of an antagonistic anti-CD40 antibody will also be given to therecipient about 8 days before transplantation and 7 and 14 days aftertransplantation. The dose will be at least about 30 mg anti-CD40antibody per kg recipient body weight.

The recipient will receive the transplant. The transplant will be cells,tissues, and/or organ from non-human animals, including but not limitedto ungulates.

For example, islet cells will be extracted from unmodified ungulates andtransplanted into human recipients suffering from diabetes. Because therecipient has been properly tolerized before transplantation, the humanrecipients will not reject the transplant.

Example 17: Treating Diabetes by Transplanting Porcine Islets in MonkeysReceiving Anti-CD40 Antibody Treatment

This example compared the effects of anti-CD40 antibody administered atdifferent time points on immuno-rejection in monkeys transplantedporcine islets.

A control diabetic monkey was transplanted with non-genetically modifiedporcine islets (FIG. 22A). The monkey was given anti-CD40 antibody onthe day of transplantation. Exogenous insulin (shown in gray bars)needed to maintain normal blood glucose level was reduced on the day oftransplant and completely stopped on day 21. Blood glucose levels (shownin lines) became normal immediately after transplantation and continueto be normal despite discontinuation of insulin on day 21 in bothmonkeys. However, the blood glucose level went after day 100 andexogenous insulin is needed to maintain normal blood glucose level afterday 125.

Porcine islets collected from wild-type pigs were transplanted to adiabetic monkey. After transplantation, a monkey was given anti-CD40antibody treatment four times through day 14 after transplantation (FIG.22B). Exogenous insulin (shown in gray bars) needed to maintain normalblood glucose level was reduced on the day of transplant and completelystopped on day 21. Blood glucose levels (shown in lines) became normalimmediately after transplantation and continue to be normal despitediscontinuation of insulin on day 21 in the monkey. The blood glucoselevel remained normal without exogenous insulin on day 250 (FIG. 22B)after transplantation.

Example 18: Immunotolerizing Diabetic Monkeys Transplanted with MonkeyIslets by Antibodies and ECDI-Fixed Splenocytes

This example compared the effects of anti-CD40 antibodies and tolerizingvaccines on immuno-rejection to allografts in a monkey (ID #13CP7). Theresults showed both the anti-CD40 antibodies and tolerizing vaccineseffectively reduced the immuno-rejection in the monkey transplanted withmonkey islets.

A diabetic monkey was transplanted with monkey islets. The monkey wasgiven an anti-CD40 antibody and rapamycin for 21 days starting from theday of transplantation. The monkey was given exogenous insulin up to 21days after transplantation. After day 21, the monkey had normal bloodglucose level in the morning (fasting), but high blood glucose level inthe afternoon (FIG. 23A). FIG. 23B demonstrates serum porcine C-peptidelevels (fasted, random, and stimulated) in the same recipient (ID#13CP7).

Example 19: Immunotolerizing Diabetic Monkeys Transplanted with PorcineIslets by α-CD40 Antibodies and CTLA4-Ig

Experiments in this example compared the effects of α-CD40 antibodiesand CTLA4-Ig on maintaining immunosuppression induced by other drugs inmonkeys transplanted with porcine islet cells. The results showed thatthe α-CD40 antibodies outperformed CTLA4-Ig in extending islet xenograftsurvival (Table 12).

Two groups of cynomolgus monkeys with streptozotocin-induced diabetes(MX-LISA-A (4 monkeys) and MX-LISA-B (3 monkeys)) were intraportallytransplanted with non-genetically modified porcine islets. For monkeysin the MX-LISA-A group, immunosuppression was induced by an α-CD25antibody, an α-CD40 antibody, sTNFR, and an α-IL-6R antibody, andmaintained by CTLA4-Ig and Rapamycin. For monkeys in the MX-LISA-Bgroup, immunosuppression was induced by an α-CD25 antibody, CTLA4-Ig,sTNFR, and an α-IL-6R antibody, and maintained by an α-CD40 antibody andRapamycin. Longer islet xenograft survival was achieved when theimmunosuppression was maintained by the α-CD40 antibody (the MX-LISA-Bgroup) compared to the MX-LISA-A group (Table 12).

TABLE 12 Immunotolerizing diabetic monkeys transplanted with porcineislets by α-CD40 antibodies and CTLA4-Ig. Islet ECDI-fixed XenograftDonor Immunosuppression Survival Group n Splenocytes InductionMaintenance (Days) MX- 4 None α-CD25 + CTLA4-Ig + 77, 126, LISA-Aα-CD40 + Rapamycin 135, 363 sTNFR + α-IL-6R MX- 3 None α-CD25 + α-CD40 +≥364, ≥365, LISA-B CTLA4-Ig + Rapamycin ≥365 sTNFR + α-IL-6R

Example 20: Immunotolerizing Diabetic Monkeys Transplanted with PorcineIslets by α-CD40 Antibodies and ECDI-Fixed Donor Splenocytes

This example examined the effects of apoptotic splenocytes onimmuno-rejection in monkeys transplanted with porcine islets. Theresults showed that the apoptotic splenocytes extended islet xenograftsurvival (Table 13).

Two groups of cynomolgus monkeys with streptozotocin-induced diabetes(MX-ECDI-Control (2 monkeys) and MX-ECDI-Vaccine (3 monkeys)) wereintraportally transplanted with non-genetically modified porcine islets.All of the monkeys were given an α-CD20 antibody, an α-CD40 antibody,sTNFR, an α-IL-6R antibody, and rapamycin from the day oftransplantation through day 21 after the transplantation. Monkeys in theMX-ECDI-Vaccine group were also given peritransplant intravenousinfusions of 0.25×10⁹ per kg bodyweight apoptotic donor splenocytes 7days before and 1 day after the transplantation. The splenocytes includethose prepared from GGTA1 knockout pigs, and those infused under thecover of the αGal glycoconjugate GAS914, as described in Katapodis etal., J Clin Invest. 110(12):1869-187 (2002), which is incorporated byreference herein in its entirety. Prolonged islet xenograft survival wasachieved in monkeys given apoptotic donor splenocytes under the cover oftransient immunosuppression (MX-ECDI Vaccine) but not in recipientsgiven transient immunosuppression only (MX-ECDI Control) (Table 13).

TABLE 13 Immunotolerizing diabetic monkeys transplanted with porcineislets by α-CD40 antibodies and apoptotic donor spenocytes IsletECDI-fixed Transient Xenograft Donor Immuno- Survival Group nSplenocytes suppression (Days) MX-ECDI- 2 None α-CD40 + α-CD20 + 32, 40Control sTNFR + α-IL-6R + Rapa Thru Day 21 MX-ECDI- 3 0.25 × 10⁹ onα-CD40 + α-CD20 + 81, 100, 113 Vaccine days −7 and +1 sTNFR + α-IL-6R +Rapa Thru Day 21

Example 21: Suppression of Circulating Immune Cells Levels by ECDI-FixedDonor Splenocytes and α-CD40 Antibodies

Experiments in this example examined ECDI-fixed cells (tolerizingvaccines) and α-CD40 antibodies on the level of circulating immune cellsafter transplantation. The levels of circulating immune cells wereindicators of transplant rejection. The results showed that bothECDI-fixed cells (tolerizing vaccines) and α-CD40 antibodies decreasedthe levels of circulating immune cells in the recipients aftertransplantation.

The circulating immune cells tested here were CD8+CD2hi CD28− effectormemory T cells, CD4+CD25hi FoxP3+CD127low regulatory T cells, andCD8+CD122+ natural suppressor cells.

CD8+CD2hi CD28− Effector Memory T Cells

Cynomolgus monkeys were transplanted with porcine islets. No tolerizingvaccine was given to the monkeys. The level of circulating CD8+CD2hiCD28− effector memory T cells was determined by flow cytometry (FIG.24). The results showed that the level of circulating CD8+CD2hi CD28−effector memory T cells in the monkeys undergoing transplantation(14GP04) was increased compared with baseline control (13CP04), and theCD8+CD2hi CD28− effector memory T cells have high prevalence within theCD8+ T cell compartment in liver mononuclear cells at the time ofsacrifice (FIG. 24).

Circulating CD8+CD2hi CD28− effector memory T cells in the two groups ofcynomolgus monkeys (MX-ECDI-control and MX-ECDI-vaccine) transplantedwith porcine islets in Example 24 were measured by flow cytometry.Monkeys in the MX-ECDI-vaccine groups received peritransplant infusionof apoptotic donor splenocytes as a tolerizing vaccine. The level ofcirculating CD8+CD2hi CD28− effector memory T cells was determined byflow cytometry (FIG. 25). Flow cytometry results show that theperitransplant infusion of apoptotic donor splenocytes (MX-ECDI-vaccine)reduced at least temporarily the posttransplant increase of circulatingCD8+CD2hi CD28− effector memory T cells in the cynomolgus monkeyscompared with control recipients that did not receive tolerizingvaccination with apoptotic donor splenocytes (MX-ECDI-control). At thetime of sacrifice (after presumed rejection), the percentage ofCD8+CD8+CD2hi CD28-effector memory T cells within the CD8+ T cellcompartment in liver mononuclear cells was comparably high in bothgroups of recipients (FIG. 25).

Circulating CD8+CD2hi CD28− effector memory T cells in monkeystransplanted with porcine islets in Examples 28 (MX-LISA-A andMX-LISA-B) and Example 29 (MX-ECDI-control and MX-ECDI-vaccine) weremeasured by flow cytometry on the day of transplantation, day 7, day 50,and day 100 after transplantation. The level of circulating CD8+CD2hiCD28− effector memory T cells from naïve monkeys was used as a control.

Flow cytometry results show that the peritransplant infusion ofapoptotic donor splenocytes (MX-ECDI vaccine) suppresses at leasttemporarily the posttransplant increase of circulating CD8+CD2hi CD28−effector memory T cells in cynomolgus monkeys compared with controlrecipients that did not receive tolerizing vaccination with apoptoticdonor splenocytes (MX-ECDI Control). The level of suppression ofposttransplant increases in CD8+ effector memory T cells inMX-ECDI-vaccine recipients was comparable with the suppression inrecipients that receive more potent and more prolonged immunosuppressionafter porcine islet xenotransplantation (the MX-LISA-A and MX-LISA-Bgroups) (FIG. 26).

CD4+CD25hi FoxP3+CD127low Regulatory T Cells

The experiments in this example examined ECDI-fixed cells (tolerizingvaccines) and α-CD40 antibodies on the level of circulating CD4+CD25hiFoxP3+CD127low regulatory T cells after transplantation. The level ofcirculating CD4+CD25hi FoxP3+CD127low regulatory T cells was anindicator of transplant rejection.

Circulating CD4+CD25hi FoxP3+CD127low regulatory T cells in monkeystransplanted with porcine islets (MX-LISA-A, MX-LISA-B, MX-ECDI-control,and MX-ECDI-vaccine) were measured by flow cytometry on the day oftransplantation, day 7, day 50, and day 100 after transplantation. Thelevel of circulating CD4+CD25hi FoxP3+CD127low regulatory T cells fromnaïve monkeys was used as a control.

Flow cytometry results show that the peritransplant infusion ofapoptotic donor splenocytes (MX-ECDI-vaccine) promoted the increase incirculating CD4+CD25hi FoxP3+CD127low regulatory T cells in cynomolgusmonkeys compared with control recipients that did not receive tolerizingvaccination with apoptotic donor splenocytes (MX-ECDI-control). Theposttransplant increase in these regulatory T cells in MX-ECDI-vaccinerecipients was comparable with the increase in recipients that receivemaintenance immunosuppression with anti-CD40 antibodies and rapamycin(MX-LISA-B) after porcine islet xenotransplantation (FIG. 27).

CD8+CD122+ Natural Suppressor Cells

Circulating CD8+CD122+ natural suppressor cells in monkeys transplantedwith porcine islets (MX-LISA-A, MX-LISA-B, MX-ECDI-control, andMX-ECDI-vaccine) were measured by flow cytometry on the day oftransplantation, day 7, day 50, and day 100 after transplantation. Thelevel of circulating CD8+CD122+ Natural Suppressor Cells from naïvemonkeys was used as a control.

Flow cytometry results showing that the peritransplant infusion of donorapoptotic splenocytes (MX-ECDI-vaccine) promoted the increase incirculating CD8+CD122+ natural suppressor cells in cynomolgus monkeyscompared with control recipients that did not receive tolerizingvaccination with apoptotic donor splenocytes (MX-ECDI-control) andMX-LISA-A recipients. The posttransplant increased in these regulatory Tcells in MX-ECDI-vaccine recipients is comparable with the increase inrecipients that receive maintenance immunosuppression with anti-CD40antibodies and rapamycin (MX-LISA-B) after porcine isletxenotransplantation (FIG. 28).

Example 22: Prolonging Pig Islet Xenograft Survival in Monkeys byECDI-Fixed Cells, Rituximab, Anti-CD40 Ab 2C10 Antibody, sTNFR,Anti-IL-6R Antibody, and Rapamycin

This example shows exemplary methods for suppressing immuno-rejectionusing ECDI-fixed donor cells in combination with other immunosuppressiondrugs.

The tolerogenic efficacy of the novel, tripartite protocol includingperitransplant i) antigen delivery on ECDI-fixed cells, ii) rapamycin,rituximab, sTNFR, and anti-IL-6R antibody, and iii) anti-CD40 Ab 2C10will be studied in the setting of intraportal transplantation of adultpig islets in monkeys.

ECDI-fixed donor splenocytes will be prepared from freshly prepared,cytokine-mobilized splenic B cells from cloned porcine donors. About0.25×10⁹/kg ECDI-fixed donor splenocytes will be administered via IV tothe monkeys on day −7 (relative to same-donor islet transplant on day0). Donor spleen will be freshly obtained from cloned porcine donorsusing splenectomy. Donor spleen B cells will be ex-vivo expanded andadministered via IV infusion to the monkeys on day +1. Adult pig isletproducts (25,000 islet equivalents/kg) from cloned porcine donors,cultured for 7 days, and meeting all release criteria will be infusedintraportally on day 0 via a portal venous vascular access port.

B cell depletion will be initiated with rituximab on day −10, i.e. priorto islet transplantation and also prior to the first infusion ofECDI-fixed donor cells. Four doses of 20 mg/kg will be administered viaIV on day −10, −3, +5, and +12 to the monkeys. The monkeys will beadministered rapamycin on day −7 through day 21 post-transplant with the12 to 15 ng/ml target trough level. sTNFR will be subcutaneouslyadministered on day −6 through day +10. Additionally, anti-IL-6R will beadministered via IV on day −7, 0, 7, 14 and 21.

Monkeys will be tested to determine the efficacy of usingpharmaceutically active agents together with ECDI-fixed donor cells in axenotransplant animal model. Three doses of 50 mg/kg anti-CD40 Ab 2C10will be administered to a monkey via IV on day −1, +7, and +14, whilefour doses of 50 mg/kg anti-CD40 Ab 2C10 will be administered to adifferent monkey via IV on day −8, −1, +7, and +14.

Post-transplant monitoring of graft functions, including daily am bloodglucose (AM BG) and pm blood glucose (PM BG), weekly C-peptide, monthlyHbA1c, and bi-monthly IVGTTs with determination of acute C-peptideresponses to glucose and glucose disappearance rates, will be measured.Successful engraftment will be defined as maintenance of nonfastingBG<200 mg/dL on greatly reduced (≤33% of baseline) or no exogenousinsulin. The primary efficacy outcome will be days to islet graftfailure as defined as the first of 3 consecutive days (on stable lowdose insulin or after discontinuation of insulin) with blood glucoselevels ≥200 mg/dL.

The islet graft function post-transplant will be further demonstrated inthe IV glucose tolerance test (IVGTT). A dose of glucose will beingested by IV and blood levels are checked at intervals. Serum porcineC-peptide responses to IV glucose before and after diabetes induction(pre and post STZ) will also be measured. A response to IV glucose atday +28 will indicate the reversal of the induced diabetic condition.

Example 23: Reducing Immuno-Rejection in a Recipient by TransplantingGenetically Modified Transplant Grafts and Administering ECDI-FixedDonor Cells

This example shows exemplary methods for suppression of immuno-rejectionin a recipient receiving a transplant from a donor by i) administeringECDI-fixed donor cells; and ii) genetically modifying the donor so thatthe transplant will induce low or no immuno-rejection in the recipient.

A human recipient in need of transplantation is tolerized to the graftby treating the recipient with ECDI fixed cells. After tolerization, therecipient will receive a transplant. The transplant will be cells,tissues, and/or organ from non-human animals, including but not limitedto ungulates. These non-human animals will be genetically modifiednon-human animals. The genetic modification will include at leastNLRC5/TAP1 knockout. Other genes that will be knocked out are listed inTables 1 and 2. Genes that will be overexpressed are listed in Tables 3and 4.

For example, a human recipient with diabetes is transplanted with one ormore NLRC5/TAP1 knockout islet cells overexpressing ICP47. Thetransplanted islet cells will overexpress a transgene coding a peptidehomologous or identical to human ICP47. The islet cells will be from agenetically modified non-human animal, such as a pig.

Following the transplantation, the human recipient will have increasedendogenous insulin levels and better glucose tolerance. When compared toa human recipient who is transplanted with wild-type islet cells, thehuman recipient transplanted with NLRC5 knockout islet cellsoverexpressing human ICP47 will have significantly reduced transplantrejection, thus requiring little to no immunosuppression therapy.

Example 24: Preventing Rejection or Extending Survival of Porcine IsletXenografts in Human Recipients in the Clinical Setting in the Absence ofChronic and Generalized Immunosuppression of the Recipients

This example shows an exemplary approach to preventing rejection orextending survival of porcine islet (and/or other cell, tissue, andorgan) xenografts in human recipients in the clinical setting in theabsence of chronic and generalized immunosuppression of the xenograftrecipient. This approach will include and integrate three components: i)genetically engineered porcine islets with deficient and/or reducedexpression of αGal, MHC class I, complement C3, and CXCL10 as well astransgenic expression the HLA-G; ii) genetically engineered donorapoptotic and non-apoptotic mononuclear cells (e.g., splenocytes) withdeficient/reduced expression of αGal, Neu5Gc, and Sda/CAD as well astransgenic expression of HLA-G with or without human CD47, human PD-L1,human PD-L2 (the genetically engineered vaccine); and iii) theadministration of transient immunosuppression including antagonisticanti-CD40 mAb, anti-CD20 mAb, rapamycin and transient anti-inflammatorytherapy including compstatin (e.g., the compstatin derivative APL-2),anti-IL-6 receptor mAb, and soluble TNF receptor.

Vaccine donor pigs comprising disrupted GGTA1, CMAH, and B4Ga1NT2 andtransgenes expressing HLA-G (or HLA-E), human CD47, human PD-L1 andhuman PD-L2 will be generated. These vaccine donor pigs will providemononuclear cells (e.g., splenocytes) with αGal-, Neu5Gc-,Sda/CAD-Deficiencies and expressing of HLA-G, human CD47, human PD-L1,and human PD-L2. Some of the mononuclear cells (e.g., splenocytes) willbe made apoptotic by ECDI fixation. Apoptotic and non-apoptoticmononuclear cells (e.g., splenocytes) will be mixed to make tolerizingvaccines. The graft donor pigs will be made by further disrupting NLRC5(or TAP1-), C3, and CXCL10 genes in the vaccine donor pigs. The graftdonor pigs will provide cells, tissues or organs (e.g., islets) fortransplant in a human recipient. The populations of vaccine donor pigsand graft donor pigs will be expanded by cloning, e.g., using somaticnuclear transfer.

A graft from the graft donor pigs will be transplanted to a recipient.Tolerizing vaccines from cells provided by the vaccine donor pigs willadministered to the human recipient one day before and 7 days aftertransplant. Immunosuppression agents such as α-CD40 antibodies, α-CD20antibodies and Rapamycin, and/or anti-inflammatory agents such ascompstatin, α-IL-6R antibodies, and sTNFR will be administered from atime point before transplant through day 21 after transplant. Thisapproach will prevent rejection or extending survival of porcinexenograft (e.g., porcine islets) in the human recipient in the absenceof chronic and generalized immunosuppression of the recipient (FIG. 5).

Example 25. Generation and Characterization of GGTA1/NLRC5 Knockout Pigs

This example shows exemplary methods for generating knockout pigs. Aknockout pig can have reduced protein expression of two or more of thefollowing: NLRC5, TAP1, C3, CXCL10, MICA, MICB, CIITA, CMAH, GGTA1and/or B4GALNT2. One of such knockout pig was a GGTA1/CMAH/NLRC5knockout pig using CRISPR/cas9 system. The pigs provided islets fortransplantation. Porcine islets with disrupted GGTA1/CMAH/NLRC5 had MHCclass I deficiency and will induce low or no immuno-rejection whentransplanted to a recipient.

Transfection of Fetal Fibroblasts

The px330 plasmids expressing guide RNA targeting GGTA1, CMAH, and NLRC5generated in Example 1 were transected in porcine fetal fibroblasts. Pigfetal fibroblasts were cultured in DMEM containing 5-10% serum,glutamine and penicillin/streptomycin. The fibroblasts wereco-transfected with two or three plasmids expressing Cas9 and sgRNAtargeting the GGTA1, CMAH or NLRC5 genes using Lipofectamine 3000 system(Life Technologies, Grand Island, N.Y.) according to the manufacturer'sinstructions.

Counter-Selection of GGTA1 KO Cells

Four days after transfection, the transfected cells were harvested andlabeled with isolectin B4 (IB4)-biotin. Cells expressing αGal werelabeled with biotin conjugated IB4 and depleted by streptavidin coatedDynabeads (Life Technologies) in a magnetic field. The αGal deficientcells were selected from the supernatant. The cells were examined bymicroscopy. The cells containing no or very few bound beads aftersorting were identified as negative cells.

DNA Sequencing Analysis of the CRISPR/Cas9 Targeted GGTA1 and NLRC5Genes

Genomic DNA from the IB4 counter-selected cells and cloned pig fetuseswere extracted using Qiagen DNeasy Miniprep Kit. PCR was performed withGGTA1 and NLRC5 specific primer pairs as shown in Table 11. DNApolymerase, dNTPack (New England Biolabs) was used and PCR conditionsfor GGTA1 were based on annealing and melting temperature ideal forthose primers. The PCR products were separated on 1% agarose gel,purified by Qiagen Gel Extraction Kit and sequenced by the Sanger method(DNA Sequencing Core Facility, University of Minnesota) with thespecific sequencing primers as shown in Table 7.

Somatic Cell Nuclear Transfer (SCNT)

SCNT was performed as described by Whitworth et al. Biology ofReproduction 91(3):78, 1-13, (2014). The SCNT was performed using invitro matured oocytes (DeSoto Biosciences Inc., St. Seymour, Tenn.).Cumulus cells were removed from the oocytes by pipetting in 0.1%hyaluronidase. Only oocytes with normal morphology and a visible polarbody were selected for SCNT. Oocytes were incubated in manipulationmedia (Ca-free NCSU-23 with 5% FBS) containing 5 μg/mL bisbenzimide and7.5 μg/mL cytochalasin B for 15 min. Oocytes were enucleated by removingthe first polar body plus metaphase II plate. A single cell was injectedinto each enucleated oocyte, fused, and activated simultaneously by twoDC pulses of 180 V for 50 μsec (BTX cell electroporator, HarvardApparatus, Hollison, Mass., USA) in 280 mM Mannitol, 0.1 mM CaCl₂, and0.05 mM MgCl₂. Activated embryos were placed back in NCSU-23 medium with0.4% bovine serum albumin (BSA) and cultured at 38.5° C., 5% CO₂ in ahumidified atmosphere for less than 1 hour, and transferred into thesurrogate pigs.

Producing Genetically Modified Pigs Using Embryos

Embryos for transferring to the surrogate pigs were added to a petridish filled with embryo transferring media. A 0.25 ml sterile straw forcell cryopreservation was also be used. Aspiration of embryos wasperformed at 25-35° C.

Aspiration of embryos was performed following this order: medialayer-air layer-media layer-air layer-embryo layer-air layer-medialayer-air layer-media layer. When the straw sterilized with EO gas wasused, its interior was washed by repeating aspiration and dispensing ofthe medium for embryo transplantation 1-3 times, before aspiration ofembryos. After the aspiration, the top end of straw was sealed by aplastic cap. To keep the aspirated and sealed straw sterile, a plasticpipette (Falcon, 2 ml) was cut in a slightly larger size than the straw,put therein, and sealed with a paraffin film. The temperature of thesealed straw was maintained using a portable incubator, until shortlybefore use.

Embryos and estrus-synchronized surrogate mothers were prepared.Transferring of embryos will be performed by exposing ovary throughlaparotomy of the surrogate mothers. After anesthetization, the mid-lineof the abdominal region was incised to expose the uterus, ovary,oviduct, and fimbriae. The straw aspirating embryos were asepticallytaken from the portable incubator, and inserted into the inlet ofoviduct. The inserted straw was moved up to the ampullary-isthmicjunction region. After the insertion procedure, the straw was cut at theair containing layer on the opposite using scissors. A 1 cc syringe wasmounted on the cut end, and approximately 0.3 cc of air was injected torelease the embryos and medium from the straw into the oviduct. At thistime, 5 mm of the top end of a 0.2 ml yellow tip was cut off and used toconnect the syringe and straw.

After the embryo transfer, the exposed uterus, ovary, oviduct, andfimbriae were put in the abdominal cavity, and the abdominal fascia wasclosed using an absorbable suture material. Then, the surgical site wascleaned with Betadine, and treated with antibiotics andanti-inflammatory and analgesic drugs. A pregnancy test of the surrogatemother transplanted with embryos was performed, followed by induction ofdelivery of non-human animals that successfully got pregnant.

Pregnancy and Fetuses

Two litters of pig fetuses (7 from pregnancy 1 and 5 from pregnancy 2)were obtained. Fetuses were harvested at day 45 (pregnancy 1) or 43(pregnancy 2) and processed for DNA and culture cell isolation. Tissuefragments and cells were plated in culture media for 2 days to allowfetal cells to adhere and grow. Wild type cells (fetal cells notgenetically modified) and fetal cells from pregnancy 1 or 2 were removedfrom culture plates and labeled with IB4 lectin conjugated to alexafluor 488 or anti-porcine MHC class I antibody conjugated to FITC. Flowcytometric analysis was performed and data shown in FIGS. 32A-32C:Pregnancy 1 or FIGS. 32D-32E: Pregnancy 2. The histogram for the WTcells are included in each panel to highlight the decrease in overallintensity of each group of fetal cells. Of specific interested is thedecrease in alpha Gal and MHC class I labeling in pregnancy 1 indicatedas a decrease in peak intensity. In pregnancy 2 fetus 1 and 3 have alarge decrease in alpha gal labeling and significant reduction in MHCclass 1 labeling as compared to WT fetal cells.

Genotypes of the Fetuses

DNA from fetal cells was subjected to PCR amplification of the GGTA1(compared to Sus scrofa breed mixed chromosome 1, Sscrofa10.2 NCBIReference Sequence: NC_010443.4) or NLRC5 (consensus sequence) targetregions and the resulting amplicons were separated on 1% agarose gels(FIGS. 29A, 29B, 30A, and 30B). Amplicons were also analyzed by sangersequencing using the forward primer alone from each reaction. Theresults are shown as Pregnancy 1 fetuses 1, 2, 4, 5, 6, and 7 truncated6 nucleotides after the target site for GGTA1. Fetus 3 was truncated 17nucleotides after the cut site followed by a 2,511 (668-3179) nucleotidedeletion followed by a single base substitution. Truncation, deletionand substitution from a single sequencing experiment containing thealleles from both copies of the target gene can only suggest a genemodification has occurred but not reveal the exact sequence for eachallele. From this analysis it appears that all 7 fetuses contained asingle allele modification. Sequence analysis of the NLRC5 target sitefor fetuses from pregnancy 1 was unable to show consistent alignmentsuggesting an unknown complication in the sequencing reaction or varyingDNA modifications between NLRC5 alleles that complicate the sangersequencing reaction and analysis. Pregnancy 2 fetal DNA samples 1, 3, 4,and 5 were truncated 3 nucleotides from the GGTA1 gene target site.Fetus 2 had variability in sanger sequencing that suggests a complexvariability in DNA mutations or poor sample quality. However, fetal DNAtemplate quality was sufficient for the generation of the GGTA1 genescreening experiment described above. NLRC5 gene amplicons were alltruncated 120 nucleotides downstream of the NLRC5 gene cut site.

Fetal DNA (from wild type (WT) controls, and fetuses 1-7 frompregnancy 1) was isolated from hind limb biopsies and the target genesNLRC5 and GGTA were amplified by PCR. PCR products were separated on 1%agarose gels and visualized by fluorescent DNA stain. The amplicon bandsin the WT lane represent unmodified DNA sequence. An increase ordecrease in size of an amplicon suggested an insertion or deletionwithin the amplicon, respectively. Variations in the DNA modificationbetween alleles in one sample might make the band appear more diffuse.Minor variations in the DNA modification were possible to resolve by a1% agarose gel. The results are shown in FIGS. 31A-31B. A lack of bandas in the NLRC5 gel (fetuses 1, 3, and 4 of Pregnancy 1; FIG. 31Abottom) suggested that the modification to the target regions wasdisrupted the binding of DNA amplification primers. The presence of allbands in GGTA1 targeting experiment suggests that DNA quality wassufficient to generate DNA amplicons in the NLRC5 targeting PCRreactions. Fetuses 1, 2, 4, and 5 of Pregnancy 1 (FIG. 31A, top) hadlarger GGTA1 amplicons, suggesting an insertion within the targetedarea. For fetus 3 of Pregnancy 1 (FIG. 31A, top), the GGTA1 ampliconmigrated faster than the WT control, suggesting a deletion within thetargeted area. For fetuses 6 and 7 of Pregnancy 1 (FIG. 31A, bottom),the NLRC5 amplicons migrated faster than the WT, suggesting a deletionwith in the target area. Fetuses 1-5 of Pregnancy 2, (FIG. 31B, top)GGTA1 amplicons were difficult to interpret by size and were diffuse ascompared to the WT control. Fetuses 1-5 (FIG. 31B, bottom) NLRC5amplicons were uniform in size and density as compared to the wild typecontrol.

Given the variation in phenotypic results for the alpha Gal and MHCclass 1 flow cytometric labeling there is considerable variation in thebi-allelic mutations in the GGTA1 and NLRC5 genes. This observation issupported by differences in band size in the agarose gels, truncatedgene products, and sequencing challenges FIGS. 29A-29B, 30A-30B,31A-31B, and 32A-32E. Cloning of individual alleles will be performed tofully decipher the sequence modifications. However, the phenotypic, DNAsequencing, and functional analysis of fetuses support the creation ofbiallelic GGTA1 and NLRC5 gene modifications in fetal pigs.

Impact of Gene Knockout on Proliferation of Human Immune Cells

Next, with cells from fetus 3 of pregnancy 1, co-culture assays wereperformed to evaluate the impact of decreased MHC class I expression onproliferation of human immune cells.

Mixed Lymphocyte Reaction (MLR)

Co-cultures were carried out in flat-bottom, 96-well plates. Human PBMCslabeled with Carboxyfluorescein succinimidyl ester (CFSE) (2.5 μM/ml),were used as responders at 0.3-0.9×10⁵ cells/well. Wild type or Porcinefibroblasts at 0.1-0.3×10⁵ cells/well (from wild type pigs or theGGTA1/NLRC5 knockout fetuses) were used as stimulators atstimulator-responder ratios of 1:1, 1:5 and 1:10. MLR co-cultures werecarried out for 4 days in all MLR assays. In another parallelexperiment, total PBMCs cells were stimulated with phytohaemagglutinin(PHA) (2 ug/ml) as positive control.

Cultured cells were washed and stained with anti-CD3 antibody, anti-CD4antibody and anti-CD8 antibody followed by formaldehyde fixation andwashed. BD FACS Canto II flow cytometer was used to assess theproliferative capacity of CD8+ and CD4+ T cells in response tofibroblasts from the GGTA1/NLRC5 knockout fetus compared to unmodifiedporcine fibroblast cells. Data were analyzed using FACS diva/Flow Josoftware (Tri star, San Diego, Calif., USA), and percentage CFSE dim/lowwas determined on pre gated CD8 T cells and CD4 T cells.

The proliferative response of human CD8+ cells and CD4+ T cells to wildtype and GGTA1/NLRC5 knockout fetal cells are shown in FIGS. 33A-33C.Cells were gated as CD4+ or CD8+ before assessment of proliferation(FIG. 33A). CD8 T cell proliferation was reduced following treatmentsstimulation by fetal cells with GGTA1/NLRC5 knockout fibroblastscompared to wild type fetal cells. Almost 55% reduction in CD8+ T cellsproliferation was observed when the human responders were treated withGGTA1/NLRC5 knockout fetal cells at 1:1 ratio (FIG. 33B). Wild typefetal cells elicited 17.2% proliferation in human CD8+ T cells whereasthe GGTA1/NLRC5 knockout fetal cells from fetus 3 (pregnancy 1) inducedonly 7.6% proliferation (FIG. 33B). No differences were observed in CD8+T cells proliferative response at 1:5 and 1:10 ratio compared to thewild type fetal cells (FIG. 33B). No changes were observed in CD4+ Tcell proliferation in response to GGTA1/NLRC5 knockout compared to thewild type fetal cells (FIG. 33C).

While some embodiments have been shown and described herein, suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the invention. It should be understood thatvarious alternatives to the embodiments of the invention describedherein will be employed in practicing the invention.

What is claimed:
 1. A genetically modified pig comprising an exogenouspolynucleotide encoding a human leukocyte antigen G (HLA-G) polypeptide,and wherein the genetically modified pig further comprises a disruptionin a gene encoding a glycoprotein galactosyltransferase alpha 1, 3(GGTA1).
 2. The genetically modified pig of claim 1, wherein the HLA-Gpolypeptide is a HLA-G1 polypeptide.
 3. The genetically modified pig ofclaim 1, wherein the exogenous polynucleotide is inserted in thegenetically modified pig's genome at a Rosa26 locus.
 4. The geneticallymodified pig of claim 1, wherein the HLA-G polypeptide has at least 80%homology to SEQ ID NO:
 53. 5. The genetically modified pig of claim 1,further comprising an exogenous nucleotide sequence encoding a humanβ2-microglobulin polypeptide, a nucleotide sequences encoding a humanleukocyte antigen E (HLA-E) polypeptide, or a combination thereof. 6.The genetically modified pig of claim 2, further comprising a disruptionin one or more genes, wherein the one or more genes encode a NOD-likereceptor family CARD domain containing 5 (NLRC5), a putative cytidinemonophosphatase-N-acetylneuraminic acid hydroxylase-like protein (CMAH),a beta-1,4-N-acetylgalactosaminyltransferase (B4GALNT2) or a combinationthereof.
 7. A genetically modified pig cell, tissue, or organ comprisingan exogenous polynucleotide encoding a human leukocyte antigen G (HLA-G)polypeptide, and wherein the genetically modified pig cell, tissue, ororgan further comprises a disruption in a gene encoding a glycoproteingalactosyltransferase alpha 1, 3 (GGTA1).
 8. A kidney, pancreas, orpancreatic tissue isolated from a genetically modified pig comprising anexogenous polynucleotide encoding a human leukocyte antigen G (HLA-G)polypeptide, and wherein the genetically modified pig further comprisesa disruption in a gene encoding a glycoprotein galactosyltransferasealpha 1, 3 (GGTA1).
 9. A method for treating a condition in a subject inneed thereof comprising: a) administering a tolerizing vaccine to thesubject; and b) transplanting a genetically modified cell, tissue, ororgan to the subject to treat the condition, wherein the geneticallymodified cell, tissue, or organ is from a donor pig comprising anexogenous polynucleotide encoding a human leukocyte antigen G (HLA-G)polypeptide, and wherein the donor pig further comprises a disruption ina gene encoding a glycoprotein galactosyltransferase alpha 1, 3 (GGTA1);wherein the condition is a diabetes, and wherein the tolerizing vaccinecomprises an apoptotic or a non-apoptotic pig cell.
 10. The method ofclaim 9, comprising transplanting the genetically modified cell, whereinthe genetically modified cell is part of a population of cells expandedex vivo or in vitro outside the donor pig prior to transplanting. 11.The method of claim 9, wherein the genetically modified cell, tissue, ororgan further comprises a disruption in one or more genes, wherein theone or more genes encode a NOD-like receptor family CARD domaincontaining 5 (NLRC5), a putative cytidinemonophosphatase-N-acetylneuraminic acid hydroxylase-like protein (CMAH),a beta-1,4-N-acetylgalactosaminyltransferase (B4GALNT2) or a combinationthereof.
 12. The method of claim 9, wherein the exogenous polynucleotideis inserted in the donor pig's genome at a Rosa26 locus.
 13. The methodof claim 9, wherein the HLA-G polypeptide has at least 80% homology toSEQ ID NO:
 53. 14. The method of claim 9, wherein the geneticallymodified cell, tissue, or organ further comprises an exogenousnucleotide sequence encoding a human β2-microglobulin polypeptide, anexogenous nucleotide sequences encoding a human leukocyte antigen E(HLA-E) polypeptide, or a combination thereof.
 15. The method of claim9, wherein the tolerizing vaccine comprises the apoptotic pig cell,wherein the apoptotic pig cell is treated with a carbodiimidederivative, and wherein the apoptotic pig cell is a leukocyte, asplenocyte, or a B-cell lymphocyte.
 16. The method of claim 9, furthercomprising administering to the subject one or more pharmaceuticalagents that inhibit T cell activation, B cell activation, dendritic cellactivation, or any combination thereof, wherein the one or morepharmaceutical agents comprise an anti-CD40 agent or an anti-CD40Lagent.
 17. A method for making a genetically modified pig comprising: a)obtaining a porcine fetal fibroblast cell comprising (i) an exogenouspolynucleotide encoding a human leukocyte antigen G (HLA-G) polypeptideor (ii) a disrupted gene encoding a glycoprotein galactosyltreansferasealpha 1,3 (GGTA1); b) genetically modifying said porcine fetalfibroblast cell using CRISPR/Cas by (i) disrupting a gene encoding GGTA1in the porcine fetal fibroblast cell comprising the exogenouspolynucleotide encoding the HLA-G polypeptide, or (ii) inserting anexogenous polynucleotide encoding an HLA-G polypeptide in the porcinefetal fibroblast cell comprising the disrupted gene encoding the GGTA1;c) transferring a nucleus of the porcine fetal fibroblast cell to aporcine enucleated oocyte to generate an embryo; and d) transferring theembryo into a surrogate pig and growing the embryo to the geneticallymodified pig in the surrogate pig.
 18. The method of claim 17, whereinthe porcine fetal fibroblast cell further comprises a disruption in oneor more genes, wherein the one or more genes encode a NOD-like receptorfamily CARD domain containing 5 (NLRC5), a putative cytidinemonophosphatase-N-acetylneuraminic acid hydroxylase-like protein (CMAH),a beta-1,4-N-acetylgalactosaminyltransferase (B4GALNT2), or acombination thereof.
 19. The method of claim 17, wherein the HLA-Gpolypeptide has at least 80% homology to SEQ ID NO:
 53. 20. The methodof claim 17, wherein the porcine fetal fibroblast cell further comprisesan exogenous nucleotide sequence encoding a human β2-microglobulinpolypeptide, an exogenous nucleotide sequences encoding a humanleukocyte antigen E (HLA-E) polypeptide, or a combination thereof. 21.The genetically modified pig of claim 1, wherein the HLA-G polypeptidecomprises at least 90% of the amino acid sequence of SEQ ID NO: 53.